Fig. 3.

PRMT5 inhibition sensitizes melanoma cells to palbociclib by suppressing CDK2 activity. (A) Western blots on cell lysates after 6-d treatment with 2 μΜ palbociclib, 500 nM (A375AR), or 250 nM (HT144AR) GSK3326595 or combination of both. (B) Proliferation curves of A375 cells expressing shp21 or shControl treated with 2 μM palbociclib. Graphs are representative of two independent experiments. Error bars represent SEM for 3 technical replicates. Shown are Western blots confirming p21 knockdown. GSK3326595 (500 nM) was used to induce p21 expression to validate the knockdown. (C) Images of colony-forming assays of A375 cells expressing shp21 or shControl treated with a combination of 1 μM palbociclib and 500 nM GSK3326595 for 14 d, and after drug removal for 14 d. Representative of 2 biological replicates with 3 technical replicates each. (D) Western blots on lysates from A375AR cells expressing shCDK2 or shControl after 24-h treatment with 2 μΜ palbociclib. (E) Proliferation curves of A375AR cells expressing shCDK2 or shControl treated with 2 μM palbociclib. Graphs are representative of 2 independent experiments. Error bars represent SEM for 3 technical replicates. (F) Western blots on total cell lysate or after immunoprecipitation of p21 complexes from A375 and A375AR after 6-d treatment with 2 μΜ palbociclib, 500 nM GSK3326595, or combination of both. (G) Western blot on melanoma cell lysates after 6-d treatment with 2 μΜ palbociclib, 250 nM (HT144AR), or 500 nM (A375AR) GSK3326595, or combination of both drugs. (H) Images of colony-forming assays of A375AR cells expressing shRB1 or shControl treated with a combination of 2 μM palbociclib and 500 nM GSK3326595 for 14 d. Representative of 2 biological replicates with 3 technical replicates each. Western blots confirm RB1 knockdown. (I) Proliferation curves of the A2058 cells treated with 2 μM palbociclib, 500 nM GSK3326595, or combination of both drugs. Error bars represent SEM for 3 technical replicates.