Extended Data Fig. 1: Evaluation of 753b-induced degradation of the BCL-2 family proteins in WI-38 cells and 753b senolytic activity against renal epithelial cells (RECs), human umbilical vein endothelial cells (HUVECs), and preadipocytes (PACs) in vitro.
A. Representative western blotting images of the levels of BCL-xL, BCL-2, BCL-w, MCL-1, and von Hippel-Lindau (VHL) in NC WI-38 cells after they were treated with increasing concentrations of 753b in a cell culture for 24 h. B. Densitometric analyses of BCL-xL, BCL-2, BCL-w, and MCL-1 expression in NC WI-38 cells from A are presented. DC50, drug concentration causing 50% degradation of protein of interest; Dmax, the maximum level of degradation of protein of interest. C. The levels of VHL, BCL-xL, BCL-2, BCL-w, and MCL-1 in NC and IR-SnC WI-38 cells and human platelets (PLTs) from three donors (P1-3) were detected by western blotting. Similar results from NC and IR-SnC WI-38 cells were observed in a separate assay. D-E. Cell viability analyses show that 753b is more potent than ABT263 against IR-SnC and REP-SnC REC (D) and HUVEC (E) but less toxic to their non-senescent counterparts. The viability of NC, IR-SnC and REP-SnC REC and HUVEC was determined 72 h after treatment with increasing concentrations of ABT263 and 753b. EC50, half-maximal effective concentration. The data presented are mean ± SD (n = 6 technical replicates) of a representative assay. EC50, half-maximal effective concentration. F & G. Cell viability analyses show that 753b is not senolytic, but dasatinib and quercetin (D + Q) are, against IR-SnC PAC. The viability of IR-SnC PAC was determined 72 h after treatment with increasing concentrations of ABT263 and 753b (F), or with vehicle (VEH), low D + Q (1 μM D plus 20 μM Q) and high D + Q (10 μM D plus 200 μM Q) (G). The data presented are mean ± SD (n = 3 technical replicates) of a representative assay. β-actin was used as a loading control in A and C.
