Figure 3. APOL1-induced cytotoxicity of high-risk human podocytes is inflammasome and STING dependent.

(A) Experimental design: Low- (G0/G0) and high-risk (G1/G2) HUPECs treated with IFN-γ. (B) Representative Western blots and (C) densitometric quantification of APOL1, NLRP3, cleaved caspase-1, STING, phosphorylated STING, and actin of G0/G0 and G1/G2 cells treated with vehicle or 2 ng/mL IFN-γ for 24 hours (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (D) Cytotoxicity, measured by LDH release, was normalized to calcein absorbance as an indicator of live cell count. G0/G0 and G1/G2 cells were treated for 24 hours with vehicle (n = 5), 0.2 ng/mL IFN-γ (n = 6), or 2 ng/mL IFN-γ (n = 6). ***P < 0.001 vs. control. (E) Cytotoxicity in G1/G2 HUPECs. G1/G2 HUPECs were treated with 2 ng/mL IFN-γ for 24 hours in the presence of inhibitors of NLRP3 (MCC950 [MCC]), caspase-1 (Ac-YVAD-CHO [Cho] and VX765 [VX]), caspase-9 (LEHD [Leh]), necroptosis (NEC1s [Nec]), ferroptosis (Liproxstatin [Lip]), p38 MAPK (SB 203580 [p38]), autophagy (choloroquine [CQ]), and inducers of autophagy (STF66247 [STF] and rapamycin [Rapa]). n = 3. ^###P < 0.001 vs. control; **P < 0.01, ***P < 0.001 vs. IFN-γ. (F) Change in intracellular Ca²⁺ (measured by FURA-2 AM fluorescence) presented as percentage change from baseline. Cells were treated with 0.2 ng/mL, 2 ng/mL, or 20 ng/mL IFN-γ for 8 hours (n = 3). *P < 0.05 vs. control-treated cells. (G) Relative calcineurin activity of G0/G0 and G1/G2 HUPECs treated with sham or the indicated concentrations of IFN-γ. **P < 0.01, ***P < 0.001 vs. control (n = 6); ^###P < 0.001 vs. indicated group. (H) Cytotoxicity of G1/G2 HUPECs. G1/G2 cells were treated for 24 hours with 2 ng/mL or 20 ng/mL IFN-γ with or without pretreatment with 0.5 μM BAPTA in Ca²⁺-free HBSS for 2 hours (n = 6). ***P < 0.001 vs. control-treated cells; red-colored, *P < 0.05 vs. indicated group. Significance was determined by 1-way ANOVA and SNK post hoc test. Data are expressed as the mean ± SEM.