Figure EV1. Prolonged CDK4/6i treatment induces p53‐dependent cellular senescence.

• A–C
  Human fibroblasts (BJ) were treated with vehicle (water for 8 times in 24 h) or abemaciclib (1 μM for 1 or 8 times in 24 h). Protein was isolated from vehicle or abemaciclib‐treated cells and immunoblotted for p‐Rb, Rb, and actin (A). RNA was isolated from treated cells, and mRNA levels of E2F2 gene (B) and p16 gene (C) were quantified by qPCR relative to tubulin (internal control). n = 3 independent experiments.
• D–F
  8‐day population doubling of WI38 cells treated with vehicle, palbociclib, or abemaciclib (both 1 μM for 8 times in 24 h; n = 3 independent experiments) (D). At 8 dpt, treated WI38 cells were incubated with EdU for 10 h and stained (scale bar, 150 μm; n = 6 samples from 3 independent experiments) (E). 3 × 103 treated WI38 cells were replated in 6‐well dish, cultured for 8 days, and stained with 0.2% crystal violet (n = 3 independent experiments) (F).
• G, H
  Cells were treated with vehicle or abemaciclib (1 μM for 1 or 4 or 8 times in 24 h), after drug withdraw, either replated for colony formation assay (n = 3 independent experiments) (G) or incubated with EdU for 10 h, and stained at 8 dpt (n = 9 samples from 3 independent experiments) (H).
• I
  Cells were treated with vehicle or abemaciclib (250 nM or 500 nM or 1 μM for 8 times in 24 h); after drug withdraw, the cells were replated for colony formation assay (n = 3 independent experiments).
• J
  Representative phase‐contrast images of BJ or WI38 cells at the end of each drug treatment (scale bar, 1 mm; n = 3 independent experiments).
• K
  At 8 dpt, treated BJ cells were fixed and stained for SA‐β‐gal and quantified (scale bar, 1 mm; n = 3 independent experiments).
• L
  Whole‐cell lysate of treated BJ cells was used to immunoblot for p16 (n = 3 independent experiments).
• M–O
  RNA‐sequencing was performed with human fibroblasts (BJ) treated with vehicle (water for 8 times in 24 h) or abemaciclib (1 μM for 8 times in 24 h) (n = 3 independent samples, sequenced together). Heatmap of cell cycle genes (M), senescence signature (N), and p53‐repressed (red) and p53‐activated (blue) cell cycle‐related genes (O) calculated from RNA‐seq datasets of cells 8 dpt relative to the vehicle‐treated group.
• P
  Cells were treated with vehicle or abemaciclib (1 μM for 8 times in 24 h); after drug withdraw, the nuclear fraction was isolated and protein extracted for Western blotting. Lamin A/C was used as the marker of nucleus and loading control (n = 3 independent experiments).

Data information: Data are means ± SD. Two‐way ANOVA (B, C, and H). One‐way ANOVA (D, E, and K). ***P < 0.001.