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Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture
利用慢病毒shRNA筛选鉴定调节上皮细胞MCF10A 3D完整性的基因   

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Abstract

MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical cell pathways that couple epithelial structure to individual cell based responses such as cell cycle exit and apoptosis. These studies will help to interrogate genetic changes critical for early breast tumorigenesis. The protocol describes a library of lentiviral shRNA constructs designed to target epithelial integrity and a highly efficient method for lentiviral transduction of suspension MCF10A cultures. Furthermore, protocols are provided for setting up MCF10A 3D cultures in Matrigel for morphometric and cellular response studies via structured illumination and confocal microscopy analysis of immunostained 3D structures.

Keywords: 3D cultures(三维培养), MCF10A(MCF10A), shRNA(shRNA), Epithelial integrity(上皮完整性), Immunofluorescence staining(免疫荧光染色), 3D imaging(三维成像), Morphometric analysis(形态分析)

Background

All epithelial cells form highly organized tissue structures, which provide physical support and a structured scaffold for coordinated cell signaling. Such coordinated signaling across the epithelial structures is fundamental for epithelial biology; enabling dynamic joint actions of epithelial cells in regulation of organ size, shape, function and individual cell based responses (Roignot et al., 2013; Shamir and Ewald, 2014). Joint command of epithelial signaling also presents a powerful tumor suppressor mechanism by gatekeeping extrinsic and intrinsic mitogenic signals to quiescent epithelial tissues (Partanen et al., 2013; Rejon et al., 2016). However, very little is still known about genetic mechanisms coupling the status of epithelial structure with individual epithelial cell functions. MCF10A 3D Matrigel culture system is a well-established genetically tractable model of mammary epithelial architecture that is widely used to explore epithelial context-dependent cell functions (Debnath and Brugge, 2005). However, individual structures in MCF10A 3D cultures are not fully uniform in size or symmetry, which makes high-throughput screens with shRNA or cDNA reagents challenging in this system. Here, we describe protocols that expand the use of MCF10A 3D culture system from single gene studies to cell pathway level perturbation studies. The protocols for medium-throughput 3D screen using validated lentiviral shRNAs were originally used in a screen designed to identify genes with epithelial integrity-linked proliferation functions (Marques et al., [2016], screen outlined in Figure 1). However, these protocols are suitable for any reverse genetic MCF10A 3D culture study within a range of about 50 perturbed genes of interest.


Figure 1. Overview of shRNA screen in MCF10A 3D culture designed to identify epithelial integrity regulating genes. The protocols described here were recently applied in a shRNA screen using 52 knockdown validated shRNAs, which were lentivirally transferred to MCF10A cells containing a switchable oncogenic form of Myc (MycERTM). This set up allowed two separate primary morphometric screens in MCF10A 3D cultures; one with and another without Myc oncogene challenge. These screens produced morphometric data from > 5,000 structures. The most interesting knockdown phenotypes were further analyzed with cell response markers (proliferation [i.e., Ki67], apoptosis [i.e., active caspase 3] and polarity change [i.e., α6-integrin, GM130]) and via 3D structures reconstructions obtained with confocal microscopy. The results from this screen for morphometric results have been published in Marques et al. (2016).

Materials and Reagents

  1. 24-well plates (VWR, catalog number: 391-3370 )
  2. Minisart filters 0.45 μm pore size (Sartorius, catalog number: 16537 )
  3. 6-well plates (VWR, catalog number: 700-1425 )
  4. 8-chamber slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 177402 )
  5. Microscope coverslips (MEDI PLAST FENNO, catalog number: 702-1051204 )
  6. Pipet tips  
  7. 500 ml bottle
  8. Ultra adherent cell culture plates for 293ft cells
    1. Nunclon surface 10 cm (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150350 )
    2. Nunclon surface 6 well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 145380 )
  9. 293ft cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: R70007 )
  10. MCF10A cells (ATCC, catalog number: CRL-10317TM )
  11. MCF10A-MycER cells (Nieminen et al., 2007)
  12. Plasmid and vectors:
    1. pCMV-dR8.91 (Delta 8.9) packaging plasmid (Marques et al., 2016)
    2. pCMV-VSVg envelope construct (Marques et al., 2016)
    3. pDSL_UGIH lentiviral shRNA vector (Alliance for Cellular Signaling) (Marques et al., 2016)
    4. pLKO lentiviral vector (Broad Institute TRC library, MISSION TRC-Hs 1.0 library) (Sigma-Aldrich) (Marques et al., 2016)
    5. pGIPZ mir-30 based vector (Open Biosystems) (Marques et al., 2016)
    6. Non-targeting shRNA control constructs (in pDSL_UGIH, pLKO.1 and pGIPZ lentiviral backbone) (Marques et al., 2016)
    7. Modified entry vector pENTR-H1 (Nieminen et al., 2007; Marques et al., 2016)
  13. Cell culture and transfection
    1. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 21041-025 )
    2. Mammary epithelial basal medium (MCDB 170) (BioConcept AG Costumer made product based on USB powder formulation [Labome, catalog number: M2162 ] without L-glutamine; catalog number: 9-02S77-I)
    3. Transfection reagent - jetPEI (Polyplus, catalog number: 101-01N )
  14. Supplements for DMEM media (added before use; See Recipes):
    1. Fetal calf serum (FCS) (Biowest, catalog number: S1810-500 )
    2. L-glutamine (Lanza, catalog number: 17-605E )
    3. Penicillin-streptomycin (Lanza, catalog number: 17-602E )
  15. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417 ) (Bionordika, catalog number: 17-516F/12 )
  16. Polybrene (Sigma-Aldrich, catalog number: H9268 )
  17. Matrigel® (Corning, catalog number: 356230 )
  18. RNeasy Mini Kit (Qiagen, catalog number: 74104 )
  19. ELB Lysis Buffer reagents (see Recipes)
    1. HEPES (Sigma-Aldrich, catalog number: H-3375 )
    2. EDTA (Sigma-Aldrich, catalog number: E5134 )
    3. Nonidet® P40 substitute (NP-40) (Sigma-Aldrich, catalog number: 74385 )
  20. Protease inhibitor cocktails
    1. Complete Mini (Roche Diagnostics, catalog number: 04693159001 )
    2. PhosphoStop (Roche Diagnostics, catalog number: 04906837001 )
  21. 0.05% trypsin (diluted from 5% trypsin-EDTA [10x]) (Thermo Fisher Scientific, GibcoTM, catalog number: 15400-054 )
  22. 4% paraformaldehyde phosphate buffer stock solution (PFA) (used as diluted 2%) (from powder: Sigma-Aldrich, catalog number: 16005-1Kg-R or from 20% aqueous solution Electron Microscopy Sciences, catalog number: 15713-S )
  23. Triton X-100, used as 0.25% dilution in PBS (Sigma-Aldrich, catalog number: 9002-93-1 )
  24. Blocking solution (see Recipes)
  25. Immunofluorescence (IF) buffer reagents (see Recipes)
    1. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S-2002 )
    2. Bovine serum albumin (BSA) (Biowest, catalog number: P6154 )
    3. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  26. Normal goat serum, used in 10% concentration (Thermo Fisher Scientific, GibcoTM, catalog number: PCN5000 )
  27. Counterstaining and Antibodies:
    1. Hoechst 33258 (Sigma-Aldrich, catalog number: 861405 )
      Note: This product has been discontinued and it has been replaced by bisBenzimide H 33258 (Sigma-Aldrich, catalog number: B2883 ).
    2. β-catenin (BD, catalog number: 610153 )
    3. GM-130 (BD, catalog number: 610823 )
    4. Anti-CD49f/α6-integrin (EMD Millipore, catalog number: CBL458 )
    5. E-cadherin (BD, catalog number: 610182 )
    6. Ki-67 (Leica Biosystems, catalog number: NCL-Ki67p )
    7. Active caspase-3 (Cell Signaling Technology, catalog number: 9661L )
    8. ZO-1 (Abcam, catalog number: ab59720 )
    9. Desmoplakin I+II (Abcam , catalog number: ab16434 or ab71690 )
    10. Desmoglein 2 (Abcam, catalog number: ab14415 )
  28. Power SYBR Green Cells-to-CT Kit (Thermo Fisher Scientific, AmbionTM, catalog number: 4402953 )
  29. Immu-MountTM reagent (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 9990412 )
  30. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-5KG-R )
  31. Supplements for MCDB 170 media (added before use; See Recipes):
    1. Bovine pituitary extract (BPE) (Sigma-Aldrich, catalog number: P-1476 ) when not available use BPE (150 mg/batch) from Upstate (Sigma-Aldrich, catalog number: 02-104 )
    2. Epithelial growth factor (EGF) (Sigma-Aldrich, catalog number: E-9644 )
    3. Transferrin (Sigma-Aldrich, catalog number: T-2252 )
    4. Isoproterenol (Sigma-Aldrich, catalog number: I-5627 )
    5. Hydrocortisone (Sigma-Aldrich, catalog number: H-4001 )
    6. Insulin (Sigma-Aldrich, catalog number: I-9278 )
    7. Amphotericin B (Sigma-Aldrich, catalog number: A-2942 )
    8. Gentamicin (Sigma-Aldrich, catalog number: G-1397 )
  32. 100 nM 4-hydroxytamoxifen used to activate MycERTM construct (4-OHT; diluted from 1 mM stock) (Sigma-Aldrich, catalog number: H7904 )
  33. SuperScript Vilo cDNA Synthesis Kit (Thermo Fisher Scientific, catalog number: 11754-050 )

Equipment

  1. 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 )
  2. Shaker (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4625Q )
  3. Confocal laser scanning Microscope (Zeiss, models: LSM Meta 510 and  780 equipped with argon [488], helium-neon [543 and 633] and diode [405] lasers and Plan-Neofluar 40x DIC objective [NA = 1.3, oil])
  4. Zeiss Axio vert 200 microscope equipped with Apotome system (Zeiss) and 20x Plan apochromat objective (NA = 0.8, air), MRr digital camera and Axiovision 4.4 software.
  5. Light Cycler 480 II instrument (Roche Diagnostics, model: Light Cycler 480 II )
  6. Nanodrop (Thermo Fisher Scientific, Thermo Scientific, model: NanoDrop 8000 )
  7. Heraeus Multifuge 3SR Plus (DJB Labcare, catalog number: 75004371 ) with Swing Swing-out rotor 4 place (DJB Labcare, catalog number: 75006445 ) and Microtitre carrier for 4 microtitre plates (DJB Labcare, catalog number: 75006449 )

Software

  1. ImageJ software (National Institute of Health, version 1.50i)

Procedure

  1. Constructing shRNA library to target putative human Epithelial Integrity Regulating genes (hEIR)
    1. shRNAs were designed to target the first and/or longest ENSEMBL transcript of each hEIR gene and 1-3 shRNA target sequences were chosen per transcript.
    2. Altogether, 136 shRNA oligos corresponding to the selected optimal target sequences (1-3 per gene) were cloned first into a pENTR cloning vector and thereafter to pDSL_UGIH lentiviral shRNA vector. Additionally 40 shRNA in pLKO lentiviral vector and 43 shRNAmirs in the pGIPZ mir-30 based vector were acquired to fully cover the previously selected target genes. Further information on the shRNA library designed to target epithelial integrity regulating genes can be found in Marques et al. (2016).
  2. Production of lentiviral particles
    1. Lentiviral vectors are transfected to 293ft cells growing in ultra adherent 24-well plates. 200,000 cells in 1 ml DMEM per well seeded one day prior transfections. Details below:
      1. Lentiviral vector mix: selected transfer plasmid (shRNA expression vector): Delta 8.9 (packaging): pCMV-VSVg (envelope) are mixed in a weight concentration ratio of 4:3:2 (tot. 10 μg; 4.44 μg, 3.33 μg, 2.22 μg) in 25 μl of 150 mM NaCl and incubated for 5 min at room temperature.
      2. Transfection: 1 μl JetPEI is mixed with 25 μl of 150 mM NaCl, and incubated for 5 min at room temperature. Lentiviral vector mix and transfection reagent JetPEI are mixed and incubated for 20 min at room temperature and dropwise added to the cells. The volume of media at the time of transfection is 0.5 ml.
    2. Transfected cells are incubated for at least 4 h at 37 °C/5% CO2 after which 0.5 ml of fresh media is added per well, making a total volume of 1 ml.
    3. After 72 h of incubation, viruses are harvested by filtering the media through a 0.45 μm filter.
    4. Occasionally viruses are produced in 6-well plates and the following formula is used: 293ft 1 million cells, 100 μl of 150 mM NaCl and 6 μl of JetPEI.
  3. MCF10A cells lentiviral infection
    1. MCF10A cells are seeded in 6-well plates aiming for a 50-60% confluence (3-4 x 105 cells per well) with 2 ml of MCDB170 + supplements media.
    2. Following day, media is changed and cells are washed with PBS. Afterwards medium and freshly harvested virus is added to cells in the ratio 1:1 (in a 6-well plate format add 500 μl viral suspension to 500 μl media).
    3. Polybrene is added to the cells at a ratio of 1:1,000 (8 μg/μl).
    4. After incubating the cells with the viruses and polybrene for 10 min at 37 °C, plates are centrifuged at 770 x g for 30 min at room temperature for virus transduction improvement.
    5. Cells are further incubated for a minimum of 4 h, after which media containing virus particles is to be replaced by 2 ml of fresh media.
  4. Cell collection for assays
    72 h post-infection MCF10A cells are either lysed for RT-qPCR and/or Western blot analysis for knockdown efficiency analysis or seeded into Matrigel® for 3D culture assays.
  5. shRNA knockdown validation
    1. RT-qPCR: Cells are lysed using the buffer supplied in the RNeasy kit and subsequent RNA isolation is performed using the kit buffers and instructions.
    2. Western blot: Cells are lysed in 40 μl of ELB lysis buffer supplemented with protease inhibitors (1/2 tablet [Complete Mini and PhosphoStop] per 10 ml of buffer).
  6. Setting up 3D culture for shRNA transduced MCF10A cells
    1. Matrigel® should be thawed overnight at 4 °C on ice.
    2. 8-chamber slides, pipets and pipet tips should be precooled at 4 °C for at least 1 h before starting the experiment.
    3. Each chamber is coated with 31 μl of Matrigel at 4 °C. Matrigel should be applied in a concentric way, avoiding contact with the chamber walls and the formation of bubbles.
    4. Matrigel-coated slides are incubated for 20-30 min at 37 °C to solidify the Matrigel.
    5. While Matrigel solidifies, shRNA transduced MCF10A growing cells should be trypsinized, counted and evaluated for viability. 1,500 viable cells are seeded per chamber on top of the Matrigel. Cells can be diluted in 20 μl to 400 μl of media, depending on the original concentration of viable cells in suspension.
    6. Media should be refreshed to the 3D cultures every third day, by carefully aspirating the old media and pipetting 400 μl of new media into the individual chambers.
  7. Fixing 3D cultured cells
    1. Media is removed and cells are washed twice with 400 μl of PBS.
    2. 200 μl of 2% PFA is used to fix the cells for 20 min at room temperature.
    3. After fixation, cells are washed twice with 400 μl of PBS at room temperature.
    4. If 3D cultures are not immunostained on the same day, cells can be stored for a couple of days (maximum time advised would be one week). This should be done at 4 °C, keeping the cells in the last PBS wash and using the 8-chamber slides lids to avoid dehydration of the samples.
  8. Immunostaining of the 3D cultures
    1. 3D cultures are permeabilized in their chambers for 10 min at room temperature using 200 μl of 0.25% Triton X-100 in PBS.
    2. Permeabilized 3D cultures are incubated at room temperature with 200 μl of blocking solution (immunofluorescence [IF] buffer with 10% normal goat serum) for 1-1.5 h.
    3. Primary antibodies are diluted in the blocking solution and added in 150 μl volume to each 3D cultures chambers (Antibodies concentration ranges from 1:50 to 1:500, see Notes for further information).
    4. Primary antibodies are incubated overnight at 4 °C using the 8-chamber slides lids.
    5. Cells are washed for three times with 400 μl of IF buffer at room temperature. Each wash performed for 20 min in a rocker at moderate speed.
    6. Afterwards cells in 3D cultures are incubated at room temperature for 45 min in 150 μl of the appropriate Alexa Fluor secondary antibody diluted in the blocking solution (In a concentration ranging from 1:150 to 1:1,000, see Notes for further information).
    7. 3D cultures are subsequently washed two times with 400 μl of IF buffer at room temperature.
    8. Nuclei are counterstained for 15 min with 150 μl of Hoechst 33258 (1:10,000 in PBS) and washed one more time with 400 μl of IF buffer at room temperature.
    9. After immunostainings, the 3D cultures are mounted by carefully removing the chamber and glue keeping them attached to the microscope slide. The coverslip is mounted with few drops of Immu-Mount reagent. 
  9. Imaging of 3D cultures
    1. The formation of MCF10A structures should be monitored with a light microscope during the 3D cultures development (every other day is advised).
    2. Images for the morphometric analysis are acquired with an inverted epifluorescence microscope coupled with apotome system equipped with a 20x Plan objective (Figure 2).
    3. Cellular response (for example, Ki67, active caspase 3) and epithelial integrity markers (for example, α6-integrin, GM130, Desmoglein, Desmoplakin ZO-1) should be studied with higher resolution imaging using confocal microscopy. For further high-resolution morphological studies and 3D reconstructions, Z-stacks can be acquired from MCF10A structures and assembled into videos and 3D deconvolutions (Videos 1 and 2).

      Video 1. Animated visualization of an acinar structure Z-stack (top to bottom). In this example the MCF10A acinar structure was grown for 10 days after knockdown of DVL3 gene. Stained for E-cadherin in red and nuclei in blue.

      Video 2. 3D structure reconstruction from the previously shown Z-stack

  10. Analyses of the images
    1. Open images using ImageJ software for morphometric analyses.
    2. Use software Image menu to convert images to 16-bit type.
    3. Image threshold should be manually adjusted (Image menu, adjust submenu). Threshold protocol allows one to enhance the binary contrast to better visualize the borders of the structures for computational quantifications. Visual side-by-side inspection of the original and thresholded images is essential to validate the authentic character of processed images (Figure 2).
    4. If the thresholding process fuses neighboring 3D structures, a Pen tool should be used to separate shapes that are obviously individual shapes in the original image.
    5. The images are analyzed for the morphometric parameters area and circularity. These and other shape descriptors can be found under analyze menu, set measurements submenu.
    6. At least 30 structures per genotype should be analyzed for the shape parameters (area and circularity). Example data are shown in Figure 3.
    7. The protocols for confocal image analyses of different cellular responses and epithelial integrity alterations are described in Marques et al. (2016).

Representative data



Figure 2. Example image before (left) and after (right) thresholding the nuclear stained image for morphometric analysis (Scale bars = 20 μm)


Figure 3. Example of the morphometric quantifications and data visualization with scatter plot. In this set of experiments, shRNA3 (purple; PARD6B shRNA) induced mild alterations in the area and circularity of the 3D acinar structures. In contrast, both shRNA1 (red; PARD6G shRNA) and shRNA2 (green; DVL3 shRNA) decreased circularity (symmetry) and increased the area (size) of the 3D acinar structures. Therefore, DVL3 and PARD6G, but not PARD6B appear to be important for the development of proper epithelial integrity.

Data analysis

  1. The morphometric values corresponding to each construct are averages of a minimum of 30 3D MCF10A structures analyzed from > 3 fields-of-view (FOV) per each experiment.
  2. The morphometric average for the control shRNA transduced structures is set to 1 and the phenotypic data can be analyzed as fold changes relative to the control.
  3. The selected thresholds were set to 20% fold change in epithelial structure area and > 10% in symmetry. For more details about the data analysis, see Marques et al. (2016).
  4. Based on the threshold values, the shRNA caused phenotypes were grouped according to the changes in size and symmetry (for example, large/hyperplastic, small & asymmetric, etc.). The results of the screen and shRNA induced phenotypes are described in Marques et al. (2016).

Notes

  1. An important methodological concern is the variability between Matrigel lots. Different lots can have dramatically different bioactivity, affecting the viability, morphology and timing of maturation (cell cycle arrest). Laboratories working with Matrigel based 3D cultures should have an internal quality control system to rigorously test each new lot for 3D structure growth supporting bioactivity for the different cell lines to be used.
  2. Primary and secondary antibody concentrations need to be tested in preliminary experiments. The ratios provided in the protocol are only guidelines.

Recipes

  1. MCDB170 supplements added per 500 ml bottle
    2.5 ml BPE from stock (200x stock - 14 mg/ml; final concentration of 70 μg/ml)
    25 μl EGF from stock (final concentration of 5 ng/ml)
    250 μl transferrin from stock (10 mg/ml 2,000x stock; final concentration of 5 μg/ml)
    1 ml isoproterenol from stock (500x stock; final concentration of 10 µM)
    250 μl hydrocortisone from stock (2,000x stock; final concentration of 0.5 μg/ml)
    250 μl insulin from stock (2,000x stock; final concentration of 5 μg/ml)
    5 ml amphotericin B from stock (50 μg/ml)
    500 μl gentamicin from stock (50 μg/ml)
  2. DMEM supplements added to 500 ml bottle
    10% FCS (50 ml from stock)
    2.5 ml L-glutamine from 2 mM stock
    1% penicillin-streptomycin (5 ml from stock)
  3. ELB lysis buffer
    150 mM NaCl
    50 mM HEPES, pH 7.4
    5 mM EDTA
    1% NP-40
  4. Immunofluorescence (IF) buffer
    7.7 mM NaN3
    0.1% bovine serum albumin
    0.2% Triton X-100
    0.05% Tween 20
    in PBS
  5. Blocking solution
    10% goat serum in IF buffer

Acknowledgments

We would like to acknowledge all the Klefström laboratory members for the critical advice and input in developing this protocol. Special thanks to lab members Johanna Englund, Topi Tervonen, Anne Makelä and Minna Ahvenainen for setting up many methods described here, Tiina Raatikainen and Tarja Välimäki for technical assistance and Eric Coles for critical comments and language accuracy. Biomedicum Imaging Unit and Biomedicum Functional Genomics Unit are acknowledged for core services and technical support. The original protocols for MCF10A 3D culture were described in Debnath et al. (2002). These protocols were modified to further expand genetic approaches (Partanen et al., 2007; Marques et al., 2016). The shRNA screen to identify epithelial regulating genes in MCF10A 3D culture has been published in Marques et al. (2016). This study was funded by the Academy of Finland, TEKES, Finnish Cancer Organizations, Helsinki Graduate Program in Biotechnology and Molecular Biology and Innovative Medicines Initiative Joint Undertaking under grant agreement No. 115188.

References

  1. Debnath, J. and Brugge, J. S. (2005). Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer 5(9): 675-688.
  2. Debnath, J., Mills, K. R., Collins, N. L., Reginato, M. J., Muthuswamy, S. K. and Brugge, J. S. (2002). The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell 111(1): 29-40.
  3. Marques, E., Englund, J. I., Tervonen, T. A., Virkunen, E., Laakso, M., Myllynen, M., Makela, A., Ahvenainen, M., Lepikhova, T., Monni, O., Hautaniemi, S. and Klefstrom, J. (2016). Par6G suppresses cell proliferation and is targeted by loss-of-function mutations in multiple cancers. Oncogene 35(11): 1386-1398.
  4. Nieminen, A. I., J. I. Partanen, A. Hau and J. Klefstrom (2007). c-Myc primed mitochondria determine cellular sensitivity to TRAIL-induced apoptosis. EMBO J 26(4): 1055-1067.
  5. Partanen, J. I., Nieminen, A. I., Makela, T. P. and Klefstrom, J. (2007). Suppression of oncogenic properties of c-Myc by LKB1-controlled epithelial organization. Proc Natl Acad Sci U S A 104(37): 14694-14699.
  6. Partanen, J. I., Tervonen, T. A. and Klefstrom, J. (2013). Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4. Philos Trans R Soc Lond B Biol Sci 368(1629): 20130111.
  7. Rejon, C., Al-Masri, M. and McCaffrey, L. (2016). Cell polarity proteins in breast cancer progression. J Cell Biochem.
  8. Roignot, J., Peng, X. and Mostov, K. (2013). Polarity in mammalian epithelial morphogenesis. Cold Spring Harb Perspect Biol 5(2).
  9. Shamir, E. R. and Ewald, A. J. (2014). Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol 15(10): 647-664.

简介

MCF10A 3D文化系统提供了腺体乳腺上皮的还原剂模型,其广泛用于研究腺体结构的发育,细胞极性和上皮完整性在上皮细胞功能的控制中的作用以及乳腺癌的机制。在这里我们描述如何使用shRNA筛选方法来识别关键细胞通路,夫妇上皮结构到个别细胞的反应,如细胞周期退出和凋亡。这些研究将有助于询问对早期乳腺肿瘤发生至关重要的遗传变化。该协议描述了设计用于靶向上皮完整性的慢病毒shRNA构建体的文库和用于悬浮MCF10A培养物的慢病毒转导的高效方法。此外,提供的协议设置MCF10A 3D文化在Matrigel的形态和细胞反应研究通过结构化照明和共聚焦显微镜分析免疫染色的三维结构。
关键字: 3D文化,MCF10A,shRNA,上皮完整性,免疫荧光染色,3D成像,形态测量分析

[背景] 上皮细胞形成高度组织的组织结构,其提供物理支持和用于协调细胞信号传导的结构化支架。跨上皮结构的这种协调的信号传导对于上皮生物学是基本的;使得上皮细胞在调节器官大小,形状,功能和基于个体细胞的应答中的动态联合作用(Roignot等人,2013; Shamir和Ewald,2014)。上皮信号传导的联合指挥还提供了一种强有力的肿瘤抑制机制,通过将外部和内部有丝分裂信号门控到静止的上皮组织(Partanen等人,2013; Rejon等人 2016)。然而,非常少有关于耦合上皮结构的状态与个体上皮细胞功能的遗传机制。 MCF10A 3D Matrigel文化系统是一个良好建立的乳腺上皮结构的基因易处理模型,广泛用于探索上皮上下文相关的细胞功能(Debnath和布鲁格,2005)。然而,MCF10A 3D培养物中的个体结构在大小或对称性上不完全均一,这使得具有shRNA或cDNA试剂的高通量筛选在该系统中具有挑战性。在这里,我们描述协议,扩大MCF10A 3D文化系统的使用从单基因研究到细胞通路水平摄动研究。使用验证的慢病毒shRNA的中等吞吐量3D屏幕的方案最初用于设计用于鉴定具有上皮完整性连接的增殖功能的基因的筛选中(Marques等人,[2016],屏幕概述于图1)。然而,这些协议适合任何反向遗传MCF10A 3D文化研究在约50扰动的感兴趣的基因范围内。


图1. MCF10A中的shRNA筛选概述设计用于鉴定上皮完整性调节基因的3D培养物。最近应用于使用52敲低验证的shRNA的shRNA筛选,其被慢病毒转移到含有可转换的致癌形式的Myc(MycER TM )的MCF10A细胞。这种设置允许在MCF10A 3D培养物中的两个单独的初级形态测定屏;一个与另一个没有Myc致癌基因挑战。这些屏幕产生从> 5,000个结构的形态数据。最有趣的敲低表型进一步用细胞应答标记物(增殖[即Ki67],凋亡[即活性胱天蛋白酶3]和极性变化[

关键字:三维培养, MCF10A, shRNA, 上皮完整性, 免疫荧光染色, 三维成像, 形态分析

材料和试剂

  1. 24孔板(VWR,目录号:391-3370)
  2. Minisart过滤器0.45μm孔径(Sartorius,目录号:16537)
  3. 6孔板(VWR,目录号:700-1425)
  4. 8室玻片(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:177402)
  5. 显微镜盖玻片(MEDI PLAST FENNO,目录号:702-1051204)
  6. 吸头提示
  7. 500 ml瓶
  8. 用于293ft细胞的超贴壁细胞培养板
    1. Nunclon表面10cm(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:150350)
    2. Nunclon表面6孔板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:145380)
  9. 293ft细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:R70007)
  10. MCF10A细胞(ATCC,目录号:CRL-10317 TM
  11. MCF10A-MycER细胞(Nieminen等人,2007)
  12. 质粒和载体:
    1. pCMV-dR8.91(Delta 8.9)包装质粒(Marques等人,2016)
    2. pCMV-VSVg包膜构建体(Marques等人,2016)
    3. pDSL_UGIH慢病毒shRNA载体(Alliance for Cellular Signaling)(Marques等人,2016)
    4. pLKO慢病毒载体(Broad Institute TRC library,MISSION TRC-Hs 1.0 library)(Sigma-Aldrich)(Marques等人,2016)
    5. 基于pGIPZ mir-30的载体(Open Biosystems)(Marques等人,2016)
    6. 非靶向shRNA对照构建体(在pDSL_UGIH,pLKO.1和pGIPZ慢病毒骨架中)(Marques等人,2016)
    7. 修饰的入门载体pENTR-H1(Nieminen等人,2007; Marques等人,2016)
  13. 细胞培养和转染
    1. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:21041-025)
    2. 乳房上皮基础培养基(MCDB 170)(BioConcept AG Costumer基于USB粉末制剂制备的产品[Labome,目录号:M2162],不含L-谷氨酰胺;目录号:9-02S77-I)
    3. 转染试剂 - jetPEI(Polyplus,目录号:101-01N)
  14. DMEM培养基补充剂(在使用前添加;参见食谱):
    1. 粪便小牛血清(FCS)(Biowest,目录号:S1810-500)
    2. L-谷氨酰胺(Lanza,目录号:17-605E)
    3. 青霉素 - 链霉素(Lanza,目录号:17-602E)
  15. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417)(Bionordika,目录号:17-516F/12)
  16. 聚凝胺(Sigma-Aldrich,目录号:H9268)
  17. Matrigel ®(Coring,目录号:356230)
  18. RNeasy Mini Kit(Qiagen,目录号:74104)
  19. ELB裂解缓冲液试剂(见配方)
    1. HEPES(Sigma-Aldrich,目录号:H-3375)
    2. EDTA(Sigma-Aldrich,目录号:E5134)
    3. Nonidet P40替代品(NP-40)(Sigma-Aldrich,目录号:74385)
  20. 蛋白酶抑制剂鸡尾酒
    1. Complete Mini(Roche Diagnostics,目录号:04693159001)
    2. PhosphoStop(Roche Diagnostics,目录号:04906837001)
  21. 0.05%胰蛋白酶(从5%胰蛋白酶-EDTA [10x]稀释)(Thermo Fisher Scientific,Gibco TM,目录号:15400-054)
  22. 4%多聚甲醛磷酸盐缓冲液储备溶液(PFA)(以粉末:Sigma-Aldrich,目录号:16005-1Kg-R或20%水溶液Electron Microscopy Sciences,目录号:15713-S使用)
  23. Triton X-100,在PBS中使用0.25%稀释(Sigma-Aldrich,目录号:9002-93-1)
  24. 阻止解决方案(参见配方)
  25. 免疫荧光(IF)缓冲液试剂(见配方)
    1. 叠氮化钠(NaN 3)(Sigma-Aldrich,目录号:S-2002)
    2. 牛血清白蛋白(BSA)(Biowest,目录号:P6154)
    3. 吐温20(Sigma-Aldrich,目录号:P9416)
  26. 以10%浓度使用的正常山羊血清(Thermo Fisher Scientific,Gibco TM ,目录号:PCN5000)
  27. 复染和抗体:
    1. Hoechst 33258(Sigma-Aldrich,目录号:861405) 注意:此产品已经停产,并已被bisBenzimide H 33258(Sigma-Aldrich,目录号:B2883)取代
    2. β-连环蛋白(BD,目录号:610153)
    3. GM-130(BD,目录号:610823)
    4. 抗CD49f /α6-整联蛋白(EMD Millipore,目录号:CBL458)
    5. E-钙粘蛋白(BD,目录号:610182)
    6. Ki-67(Leica Biosystems,目录号:NCL-Ki67p)
    7. 活性半胱天冬酶-3(Cell Signaling Technology,目录号:9661L)
    8. ZO-1(Abcam,目录号:ab59720)
    9. Desmoplakin I + II(Abcam,目录号:ab16434或ab71690)
    10. Desmoglein 2(Abcam,目录号:ab14415)
  28. Power SYBR Green Cells-to-CT Kit(Thermo Fisher Scientific,Ambion TM ,目录号:4402953)
  29. Immu-Mount TM试剂(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:9990412)
  30. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434-5KG-R)
  31. MCDB 170培养基补充剂(在使用前添加;查看食谱):
    1. 当不可获得时,使用来自Upstate(Sigma-Aldrich,目录号:02-104)的BPE(150mg /批),牛垂体提取物(BPE)(Sigma-Aldrich,目录号:P-1476)
    2. 上皮生长因子(EGF)(Sigma-Aldrich,目录号:E-9644)
    3. 转铁蛋白(Sigma-Aldrich,目录号:T-2252)
    4. 异丙肾上腺素(Sigma-Aldrich,目录号:I-5627)
    5. 氢化可的松(Sigma-Aldrich,目录号:H-4001)
    6. 胰岛素(Sigma-Aldrich,目录号:I-9278)
    7. 两性霉素B(Sigma-Aldrich,目录号:A-2942)
    8. 庆大霉素(Sigma-Aldrich,目录号:G-1397)
  32. 100nM 4-羟基他莫昔芬用于活化MycER TM sup构建体(4-OHT;从1mM储备液稀释)(Sigma-Aldrich,目录号:H7904)
  33. SuperScript Vilo cDNA合成试剂盒(Thermo Fisher Scientific,目录号:11754-050)

设备

  1. 37℃,5%CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific Inc.,型号:Forma TM supra系列II 3110)中, >
  2. Shaker(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4625Q)
  3. 共聚焦激光扫描显微镜(Zeiss,型号:配备有氩气[548],氦 - 氖[543和633]和二极管[405]激光器和Plan-Neofluar 40x DIC物镜[NA = 1.3,油]的LSM Meta 510和780)
  4. 装备有Apotome系统(Zeiss)和20x平面消色差物镜(NA = 0.8,空气),MRr数码相机和Axiovision 4.4软件的Zeiss Axio vert 200显微镜。
  5. Light Cycler 480 II仪器(Roche Diagnostics,型号:Light Cycler 480 II)
  6. Nanodrop(Thermo Fisher Scientific,Thermo Scientific,型号:NanoDrop 8000)
  7. 具有Swing Swing-out转子4位置(DJB Labcare,目录号:75006445)的Heraeus Multifuge 3SR Plus(DJB Labcare,目录号:75004371)和用于4个微量滴定板(DJB Labcare,目录号:75006449)的Microtitre载体

软件

  1. ImageJ软件(国立卫生研究院,版本1.50i)

程序

  1. 构建shRNA文库以靶向推定的人上皮完整性调节基因(hEIR)
    1. 设计shRNA以靶向每个hEIR基因的第一和/或最长ENSEMBL转录物,并且每个转录物选择1-3个shRNA靶序列。
    2. 总共,将对应于选择的最佳靶序列(每个基因1-3个)的136个shRNA寡核苷酸首先克隆到pENTR克隆载体中,然后克隆到pDSL_UGIH慢病毒shRNA载体。另外,获得pLKO慢病毒载体中的40个shRNA和基于pGIPZ mir-30的载体中的43个shRNA,以完全覆盖先前选择的靶基因。关于设计用于靶向上皮完整性调节基因的shRNA文库的进一步信息可以在Marques等人中找到。 (2016)。
  2. 慢病毒颗粒的生产
    1. 将慢病毒载体转染到在超粘附24孔板中生长的293ft细胞。在转染前一天接种每孔1ml DMEM中的200,000个细胞。详情如下:
      1. 慢病毒载体混合物:选择的转移质粒(shRNA表达载体):将Delta 8.9(包装):pCMV-VSVg(包膜)以4:3:2(总重量10μg;4.44μg,3.33μg, 2.22μg)的25μl150mM NaCl中,并在室温下温育5分钟。
      2. 转染:将1μlJetPEI与25μl150mM NaCl混合,并在室温下温育5分钟。将慢病毒载体混合物和转染试剂JetPEI混合并在室温下孵育20分钟,并滴加到细胞中。转染时的培养基体积为0.5ml
    2. 将转染的细胞在37℃/5%CO 2下孵育至少4小时,然后每孔加入0.5ml新鲜培养基,使总体积为1ml。
    3. 培养72小时后,通过0.45μm过滤器过滤培养基收集病毒。
    4. 偶尔在6孔板中产生病毒,使用以下公式:293ft 1百万个细胞,100μl的150mM NaCl和6μl的JetPEI。
  3. MCF10A细胞慢病毒感染
    1. 将MCF10A细胞接种在6孔板中,目标是用2ml MCDB170 +补充培养基进行50-60%汇合(3-4×10 5个细胞/孔)。
    2. 第二天,更换培养基并用PBS洗涤细胞。然后将中和新鲜收获的病毒以1:1的比例加入细胞(在6孔板形式中,将500μl病毒悬浮液加入500μl培养基中)。
    3. 聚凝胺以1:1,000(8μg/μl)的比例加入到细胞中
    4. 在37℃下将细胞与病毒和聚凝胺孵育10分钟后,将板在室温下以770×g离心30分钟以进行病毒转导改善。
    5. 将细胞进一步温育至少4小时,然后用2ml新鲜培养基替换含有病毒颗粒的培养基。
  4. 用于测定的细胞收集
    感染后72小时将MCF10A细胞裂解用于RT-qPCR和/或Western印迹分析以敲低效率分析或接种到用于3D培养测定的Matrigel 中。
  5. shRNA敲低验证
    1. RT-qPCR:使用RNeasy试剂盒中提供的缓冲液裂解细胞,随后使用试剂盒缓冲液和说明书进行RNA分离。
    2. 蛋白质印迹:将细胞在补充有蛋白酶抑制剂(1/2片剂[Complete Mini和PhosphoStop]/10ml缓冲液)的40μlELB裂解缓冲液中裂解。
  6. 为shRNA转导的MCF10A细胞设置3D培养物
    1. Matrigel 应在4℃冰上解冻过夜。
    2. 8小室玻片,移液管和移液管吸头应在开始实验前在4°C预冷至少1小时。
    3. 每个室在4℃下用31μl的基质胶包被。 Matrigel应以同心方式应用,避免与室壁接触和气泡的形成。
    4. 将基质胶包被的载玻片在37℃下孵育20-30分钟以固化基质胶。
    5. 当基质胶固化时,shRNA转导的MCF10A生长的细胞应当被胰蛋白酶消化,计数并评价其存活力。每个室在基质胶的顶部接种1,500个活细胞。根据悬浮液中活细胞的原始浓度,细胞可以在20μl至400μl培养基中稀释。
    6. 通过小心地吸取旧培养基并将400μl新培养基吸入各个室中,培养基应每三天更新至3D培养物。
  7. 固定3D培养细胞
    1. 除去培养基,用400μlPBS洗涤细胞两次
    2. 使用200μl的2%PFA在室温下固定细胞20分钟
    3. 固定后,在室温下用400μlPBS洗涤细胞两次
    4. 如果3D培养物在同一天没有免疫染色,细胞可以储存几天(建议的最大时间是一周)。这应该在4°C进行,保持细胞在最后一次PBS清洗和使用8室幻灯片盖,以避免样品脱水。
  8. 3D培养物的免疫染色
    1. 3D培养物在室中使用200μl的0.25%Triton X-100在PBS中在室中透化10分钟。
    2. 渗透的3D培养物在室温下与200μl封闭溶液(具有10%正常山羊血清的免疫荧光[IF]缓冲液)温育1-1.5小时。
    3. 将一抗在封闭溶液中稀释,并以150μl体积加入每个3D培养室中(抗体浓度范围为1:50至1:500,更多信息参见说明)。
    4. 使用8室玻片盖在4℃下将一级抗体温育过夜。
    5. 在室温下用400μlIF缓冲液洗涤细胞三次。每次洗涤以中等速度在摇床中进行20分钟。
    6. 之后,将3D培养物中的细胞在室温下在150μl在封闭溶液中稀释的合适的Alexa Fluor二抗(在浓度范围从1:150至1:1,000,参见注释获得进一步信息)中温育45分钟。
    7. 3D培养物随后在室温下用400μlIF缓冲液洗涤两次
    8. 将核用150μlHoechst 33258(在PBS中1:10,000)复染色15分钟,并在室温下用400μlIF缓冲液再洗涤一次。
    9. 免疫染色后,通过小心地移除腔室和胶水,保持它们附着到显微镜载玻片上来安装3D培养物。盖玻片安装有几滴Immu-Mount试剂。
  9. 3D文化的成像
    1. 在3D文化发展期间(建议每隔一天)应用光学显微镜监测MCF10A结构的形成。
    2. 形态测量分析的图像用倒置落射荧光显微镜,配备有配备20x Plan物镜(图2)的apotome系统获得。
    3. 应使用共聚焦显微镜用更高分辨率的成像研究细胞反应(例如,Ki67,活性半胱天冬酶3)和上皮完整性标记(例如,α6-整联蛋白,GM130,桥粒核心糖蛋白,桥粒蛋白ZO-1)。对于进一步的高分辨率形态学研究和3D重建,Z叠加可以从MCF10A结构获取并组装成视频和3D解卷积(视频1和2)。

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      视频1.腺泡结构Z形堆栈(从上到下)的动画可视化。 在本实施例中,MCF10A腺泡结构在敲除DVL3基因后生长10天。染色为E-钙粘蛋白的红色和细胞核的蓝色。
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      视频2.从之前显示的Z-stack的3D结构重建
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  10. 图像分析
    1. 使用ImageJ软件打开图像进行形态测定分析。
    2. 使用软件图像菜单将图像转换为16位类型。
    3. 图像阈值应手动调整(图像菜单,调整子菜单)。阈值协议允许增强二进制对比度以更好地可视化用于计算量化的结构的边界。对原始和阈值图像的可视并排检查对于验证处理图像的真实性质是必不可少的(图2)。
    4. 如果阈值处理过程融合相邻的3D结构,则应当使用Pen工具来分离原始图像中明显是各个形状的形状。
    5. 分析图像的形态测量参数面积和圆形度。这些和其他形状描述符可以在分析菜单,设置测量子菜单下找到
    6. 每个基因型至少30个结构应分析形状参数(面积和圆形度)。示例数据如图3所示。
    7. 不同细胞反应和上皮完整性改变的共聚焦图像分析的方案描述于Marques等人。 (2016年)。

代表数据



图2.用于形态测量分析的核染色图像(比例尺=20μm)之前(左)和之后(右)的示例图像


图3.在这组实验中,shRNA3(紫色; PARD6B shRNA)诱导了3D腺泡结构的面积和圆形度的轻微改变。相比之下,shRNA1(红色; PARD6G shRNA)和shRNA2(绿色; DVL3 shRNA)减少圆形(对称性)和增加面积(大小)的3D腺泡结构。因此,DVL3和PARD6G,但不是PARD6B似乎对于正确的上皮完整性的发展是重要的。

数据分析

  1. 对应于每个构建体的形态测定值是从>分析的最小30个3D MCF10A结构的平均值。 3视野(FOV)。
  2. 对照shRNA转导结构的形态平均值设置为1,并且表型数据可以作为相对于对照的倍数变化来分析。
  3. 选择的阈值设定为上皮结构面积的20%倍变化, 10%对称。有关数据分析的更多详情,请参阅Marques 。 (2016)。
  4. 基于阈值,根据大小和对称性的变化(例如,大/增生,小&不对称,等)将shRNA引起的表型分组。筛选和shRNA诱导的表型的结果描述于Marques等人。 (2016年)。

笔记

  1. 一个重要的方法问题是基质胶批次之间的变异性。不同批次可具有显着不同的生物活性,影响成活的可行性,形态和时间(细胞周期停滞)。实验室使用基于matrigel的3D文化应该有一个内部质量控制系统,严格测试每个新的批次为3D结构增长支持生物活性的不同细胞系使用。
  2. 初级和二级抗体浓度需要在初步实验中测试。协议中提供的比率仅为指导原则。

食谱

  1. 每500 ml瓶子添加MCDB170补充剂。
    2.5ml来自原液的BPE(200x原液 - 14mg/ml;终浓度为70μg/ml) 25μl来自库存的EGF(终浓度为5ng/ml)
    250μl来自储备液的转铁蛋白(10mg/ml 2,000x储液;最终浓度5μg/ml)
    1ml来自储备的异丙基肾上腺素(500×储备液;终浓度为10μM) 250μl来自储备液的氢化可的松(2,000x储备液;终浓度为0.5μg/ml)
    250μl来自库存的胰岛素(2,000x储备液;终浓度5μg/ml)
    5ml来自储备液的两性霉素B(50μg/ml)
    500μl来自原液(50μg/ml)的庆大霉素
  2. DMEM补充剂添加到500 ml瓶
    10%FCS(50 ml,从库存)
    来自2mM原液的2.5ml L-谷氨酰胺
    1%青霉素 - 链霉素(5ml,来自库存)
  3. ELB裂解缓冲液
    150mM NaCl 50mM HEPES,pH7.4 5 mM EDTA
    1%NP-40
  4. 免疫荧光(IF)缓冲液
    7.7 mM NaN 3
  5. 封锁解决方案
    10%山羊血清在IF缓冲液中

致谢

我们要感谢所有Klefström实验室成员为所有的关键建议和输入在开发这个协议。特别感谢实验室成员Johanna Englund,Topi Tervonen,AnneMakelä和Minna Ahvenainen设置了这里描述的许多方法,Tiina Raatikainen和TarjaVälimäki的技术援助和Eric Coles的批评性评论和语言准确性。生物医学成像单元和生物医学功能基因组单元被承认用于核心服务和技术支持。 MCF10A 3D培养物的原始方案描述于Debnath等人的中。 (2002)。修改这些方案以进一步扩展遗传方法(Partanen等人,2007; Marques等人,2016)。在MCF10A 3D培养物中鉴定上皮调节基因的shRNA筛选已经在Marques等人中公开。 (2016年)。这项研究由芬兰科学院,TEKES,芬兰癌症组织,赫尔辛基生物技术和分子生物学研究生课程和创新药物倡议联合承诺根据授权协议第115188号资助。

参考文献

  1. Debnath,J.和Brugge,JS(2005)。  在三维培养物中建模腺上皮癌。 Nat Rev Cancer 5(9):675-688。
  2. Debnath,J.,Mills,KR,Collins,NL,Reginato,MJ,Muthuswamy,SK和Brugge,JS(2002)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/12372298"target ="_ blank">细胞凋亡在正常和癌基因表达的乳腺腺泡内产生和维持管腔空间的作用细胞111(1 ):29-40。
  3. Marck,E.,Englund,JI,Tervonen,TA,Virkunen,E.,Laakso,M.,Myllynen,M.,Makela,A.,Ahvenainen,M.,Lepikhova,T.,Monni,O.,Hautaniemi, S.和Klefstrom,J.(2016)。  Par6G抑制细胞增殖并且被多种癌症中的功能缺失突变所靶向。致癌基因35(11):1386-1398。
  4. Nieminen,AI,JI Partanen,A.Hau和J. Klefstrom(2007)。  c-Myc引发的线粒体决定细胞对TRAIL诱导的细胞凋亡的敏感性。 EMBO J 26(4):1055-1067。
  5. Partanen,JI,Nieminen,AI,Makela,TP and Klefstrom,J。(2007)。  通过LKB1控制的上皮组织抑制c-Myc的致癌性质。 Proc Natl Acad Sci USA 104(37):14694-14699。 />
  6. Partialen,JI,Tervonen,TA和Klefstrom,J.(2013)。  在癌症中破坏上皮极性屏障:LKB1/PAR-4的奇怪病例。 Philos Trans R Soc Lond B Biol Sci 368(1629):20130111.
  7. Rejon,C.,Al-Masri,M.和McCaffrey,L.(2016)。  乳腺癌进展中的细胞极性蛋白。 J Cell Biochem 。
  8. Roignot,J.,Peng,X.和Mostov,K.(2013)。  极性在哺乳动物上皮形态发生。 Cold Spring Harb Perspect Biol 5(2)
  9. Shamir,ER和Ewald,AJ(2014)。  三 - 三维器官培养:哺乳动物生物学和疾病的实验模型。 Nat Rev Mol Cell Biol 15(10):647-664。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Marques, E. and Klefström, J. (2016). Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture. Bio-protocol 6(23): e2050. DOI: 10.21769/BioProtoc.2050.
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