Fig. 3: CDK4/6 inhibition induces SASP-mediated EGFR activation.

A, B Pull down assay of GTP-KRAS in MIA PaCa-2-RB7LP cells or MIA PaCa-2 cells treated with or without 1 μg/mL DOX or 10 µM palbociclib for the indicated time. C IB of the indicated proteins in MIA PaCa-2 cells treated with or without 10 µM palbociclib for 0-72 h. D Representative immunofluorescence (IF) staining of phospho-EGFR in MIA PaCa-2 cells treated with or without 10 µM palbociclib for 72 h. Scale bars, 50 µm. E Enrichment plots of EGFR_UP.V1_UP pathway from GSEA (MIA PaCa-2 cells). F, G RT-qPCR determination of EGF family of ligands and Sprouty family members in MIA PaCa-2 cells treated as in (C). H IB of the indicated proteins in MIA PaCa-2 cells pre-treated with or without 10 µM palbociclib for 48 h thereafter with the indicated doses of JSH-23 for 48 h. I IHC of Ki-67 in MIA PaCa-2 cells pre-treated with or without 10 µM palbociclib for 48 h thereafter with the indicated doses of JSH-23 for 48 h. Scale bars, 20 µm. J Representative images of SA-β-gal staining in the indicated cells pre-treated with or without 10 µM palbociclib for 48 h, thereafter with 30 µM JSH-23 for 48 h. Scale bars, 100 µm. Quantitation of SA-β-gal positive cells from 3 or more randomly chosen fields. K RT-qPCR determination of TGF-α and Amphiregulin in the indicated cells pre-treated with or without 10 µM palbociclib for 48 h, thereafter with 30 µM JSH-23 for 48 h. α-tubulin was used as a loading control. DMSO was used as a vehicle. All data are presented as mean ± SD of three independent experiments. One-way ANOVA followed by Tukey’s post-hoc test was performed in (F, G, J and K). *p < 0.05, **p < 0.01, ***p < 0.001.