Figure 4.

ITGB3 Regulates Senescence Independently of Its Binding Activity

(A) Endogenous αvβ3 expression increases during OIS upon RAS expression in BFs. Representative pictures for αvβ3 (green) and F-actin (red) staining by IF in vector and RAS cells are shown. αvβ3-stained FA complexes are indicated with white arrows. Data represent the percentage of cells positive for αvβ3 staining. Scale bar, 20 μm.

(B) ITGB3 is endogenously upregulated during DNA-damage-induced senescence (DDIS). BFs were treated with 100 μM etoposide (Etop) for 2 days and replaced with fresh media for 5 days.

(C) MCF7 breast cancer cells were treated with 200 nM palbociclib (Palbo) for 7 days, after which cells were lysed for immunobloting. An increase in β3 subunit and p53 can be observed after Palbo treatment.

(D) mRNA analyses for ITGB subunits 1–8 during RAS-induced senescence in BFs.

(E) CBX7 binding to ITGB3 TSS is reduced during OIS. ChIP for CBX7 enrichment (black bars) versus IgG control (white bars) at an ITGB3 TSS and a coding region in BFs expressing vector or RAS.

(F) Schematic representation of the timings used to determine the role for ITGB3 overcoming OIS (top panel). Lower left panel: relative cell number in RAS-expressing BFs transduced with a vector or an shRNA targeting ITGB3 (shITGB3). Lower right panel: representative immunoblot showing β3 subunit knockdown efficiency.

(G) Top panel shows the experimental planning. Senescence was induced by Palbo treatment, after which siITGB3 was transfected (green bar). Two independent siRNAs targeting ITGB3 (siITGB3) overcame the senescence arrest induced by treating MCF7 cells with Palbo for 7 days. BrdU was added 24 hr before the end of the experiment.

(H) Cells expressing RAS were treated with DMSO or αvβ3 inhibitor (cilengitide) for 48 hr, and the relative cell number was calculated. Increasing concentrations of cilengitide (10, 25, and 50 nM) show no reversion of the proliferation arrest induced by RAS. An inhibitor for TGF-β-receptor 1 (TGFBR1, 4 μM) was used as positive control.

(I) Immunoblot for the conditioned media (CM) from cells expressing either vector or RAS treated with or without 50 nM of αvβ3 inhibitor (cilengitide) for 48 hr, followed by a 72-hr incubation in fresh media. Coomassie staining is shown as loading control.

^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.