Fig 1: CDK4/6is promote PARP1 protein degradation and regulate DNA repair factor availability and DNA repair competency in RB-proficient NSCLC cells. (A) Western blot detection of PARP1 levels in A549 cells treated with palbociclib (P, 1 µM) or abemaciclib (A, 1 µM) for the indicated times. (B) Western blot detection of PARP1 levels in A549 cells treated with increasing concentrations of palbociclib (P) for 24 h (top) and with P (1 µM) for the indicated times (bottom). (C) Co-treatment with the proteasome inhibitor MG132 inhibits CDK4/6i-induced PARP1 degradation. A549 cells were treated with palbociclib (1 µM) for 16 h and MG132 for an additional 6 h PARP1 and PAR in total cell lysates were analyzed by Western blot. (D) Western blot detection of PARP1 levels in A549 cells treated with control or CDK4/6i (palbociclib or abemaciclib, 1 μM) in the presence of cycloheximide (CHX; 10 μg /ml) for the indicated times. (E) Western blot detection of PARylation levels in A549 cells treated with 1 μM palbociclib or abemaciclib for 2 h, followed by MMS (0.01 %) for an additional 1 or 3 h (F) Representative images (left) of γH2AX (red) and RAD51 (green) staining in A549 cells treated with vehicle (V, DMSO), palbociclib (P, 1 μM) for 16 h and with/without IR (2Gy) for additional 4 h Cells with white squares are enlarged below for a better visualization. The arrows indicate the position of the foci. The box and whisker plot (right) quantifies cells with more than 5 foci. *P < 0.01 V+IR vs. P+IR, by 1-way ANOVA with Tukey's multiple comparisons test. Scale bar, 40 μm.