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. 2017 Jul 3;36(17):2510–2528. doi: 10.15252/embj.201696089

Figure EV2. Inactivation of Skp2 does not effectively suppress YAP‐driven cell cycle control in a 2D culture system.

Figure EV2

  1. Using the CRISPR‐Cas9 system, we generated control or Skp2 knockout MCF10A clones. These were further manipulated to generate the doxycycline‐inducible 5SA‐YAP expression system (Tet‐ON: 5SA‐YAP). Immunoblots confirmed both the Skp2 knockout and the function of the inducible 5SA‐YAP expression system.
  2. (Above) Experimental protocol schematic. (Below) Cells prepared as in (A) were treated with or without doxycycline for 2 days. Then, cells were either serum‐starved or not in the absence or presence of 2 days of doxycycline. After 1 h of BrdU incorporation, the BrdU‐positive cells were counted. > 500 cells were analyzed in each of three independent experiments.
  3. (Above) Experimental protocol schematic. Doxycycline‐inducible 5SA‐YAP‐expressing MCF10A cells were either serum‐starved or not. After 2 days, some cells were fixed and others were treated with doxycycline. After an additional 2 days, the cells were fixed and stained with a Ki67‐specific antibody and DAPI.
  4. Immunoblots of cells treated as in (C).
  5. Quantification of Ki67 positivity among cells treated as in (C). > 300 cells were analyzed for each of two independent experiments.
  6. Doxycycline‐inducible 5SA‐YAP‐expressing MDA‐MB‐231 cells treated as in (C) except for an additional 1 h of BrdU incorporation. > 300 cells were analyzed for each of two independent experiments.
  7. MCF10A cells were infected with lentiviruses encoding either vector or Skp2. The cells were then serum‐starved for 2 days to induce cell cycle exit. After 1 h BrdU incorporation, the cells were fixed and stained with BrdU‐ or Ki67‐specific antibodies. (Left) Representative cells stained for BrdU and counterstained with DAPI. Scale bars: 50 μm. (Right) Quantification of BrdU‐ or Ki67‐positive cells. > 800 cells were analyzed in each of three independent experiments.
  8. RPE1 cells were treated with DMSO or SZL P1‐41. After 48 h, cells were fixed and stained with a p27‐specific antibody and DAPI. Scale bars: 20 μm.
  9. (Left) Experimental protocol schematic. Doxycycline‐inducible 5SA‐YAP‐expressing cells were grown on 3D Matrigel for acinus formation. After arresting acinar growth 8 days later, one group was fixed and the other two were treated with or without doxycycline for 3 days before also being fixed. (Right) All fixed samples were visualized and quantified using phase contrast microscopy and the ImageJ program. Orange bars indicate the median. > 200 acini were analyzed for each of two independent experiments.
Data information: All error bars indicate s.e.m. Statistical significance, as determined by a two‐tailed t‐test, is indicated in the graph. N.S. indicates non‐significance.