Figure 4. Signaling through ITGB4, mTORC2, and AKT is required for a senescence program triggered by FLRT2 depletion.

(A and B) HUVECs were transfected with Con Si, ITGB1 Si, ITGB3 Si, ITGB4 Si, or ITGB5 Si (first transfection) 6 hours before transfection with Con Si or FLRT2 Si (second transfection). At day 2 after transfection, cells were harvested and subjected to immunoblotting (A). At day 3 after transfection, SA-β-Gal activity was examined (B). (C) HUVECs were transfected with Con Si or FLRT2 Si, and reverse transcription (RT) followed by real-time quantitative polymerase chain reaction (qPCR) (RT-qPCR) analysis was carried out 2 days after transfection. (D and E) HUVECs were transfected with Con Si or FLRT2 Si, followed by treatment with an inhibitor of ITGB4 (ASC-8). Immunoblot assay (D) and SA-β-Gal assay (E) were performed at days 2 and 3 after transfection, respectively. Scale bar: 10 μm. (F) HUVECs were transfected with Con Si or ITGB4 Si 1 day before transfection with empty vector (EV), a vector encoding wild-type ITGB4 (WT), or a vector encoding a truncated mutant of ITGB4 lacking the residues downstream of amino acid 1355 (ΔCYT), followed by transfection with Con Si or FLRT2 Si. At day 2 after transfection, the cells were subjected to immunoblotting analysis. (G) HUVECs were transfected with Con Si or ITGB4 Si. At 6 hours after transfection, one-half of each group was transfected with Con Si, while the other half was transfected with FLRT2 Si. At day 2 after transfection, cell lysates were subjected to immunoprecipitation with anti-Rictor antibody. Mean ± SD (n = 3; ^#P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001). One-way ANOVA.