Figure EV3. HDAC3 knockdown undermines AKT phosphorylation and prostate cancer cell growth in 3D culture.

• A
  C4‐2 cells stably infected with lentivirus for control or HDAC3‐specific shRNAs were harvested for Western blots with the indicated antibodies.
• B
  C4‐2 cells were infected with lentivirus as in (A) and subjected to cell growth analysis using a 2D cell culture system. The cell growth was measured in triplicate for each of three independent experiments. Data are shown as means ± SEM. shC versus shHDAC3‐#1: ***P = 6.0e‐04, shC versus shHDAC3‐#2: ***P = 6.5e‐04, shC versus shHDAC3‐#3: **P = 0.0012 comparing the cell growth at day 4 by the unpaired two‐tailed Student's t‐test.
• C
  An illustration shows the workflow of a 3D Matrigel culture system.
• D, E
  C4‐2 cells were infected with lentivirus as in (A) and subjected to cell growth analysis using a 3D cell culture system. The representative images from 3D cultures at day 5 are shown in (D) with quantitative data in a box plot (E). Scale bar, 100 μm. In graph (E), the description for box plot is the same as the figure legend in Fig 7H. Data are shown as means ± SEM. shC (n = 321) versus shHDAC3‐#1 (n = 397): ***P = 2.02e‐31, shC (n = 321) versus shHDAC3‐#2 (n = 402): ***P = 4.43e‐54, shC (n = 321) versus shHDAC3‐#3 (n = 334): ***P = 4.35e‐06 were performed by Wilcoxon rank sum test with continuity correction.
• F, G
  C4‐2 cells in 3D cultures at day 5 were subjected for IFC analysis for expression of total and S473 phosphorylated AKT. The representative images of AKT phosphorylation (S473) are shown in (F) with quantitative data in (G). Scale bar for images in (F), 20 μm.

Source data are available online for this figure.