Loss of VPS13C results in activation of the cGAS - STING pathway. (A) Cartoon schematic of cGAS-STING signaling pathway and STING trafficking through the Golgi to lysosomes for degradation. (B) qPCR of five ISG transcripts (IFIT1, IFIT3, ISG15, OASL, and STAT1) shows increased expression in VPS13C^KO HeLa cells. n = 3 biological replicates. Created with BioRender.com. (C) qPCR of three ISG transcripts after treatment with siRNA against cGAS (top row) or STING (bottom row). n = 4 biological replicates. (D) IB showing efficiency of STING depletion after treatment with anti-STING siRNA. (E) IB showing increased levels of phosphorylated STING, TBK1, and IRF3, indicating activation of the cGAS-STING pathway. Note that the upper band in lanes 2 and 3 of the anti-STING blot corresponds to p-STING (lanes 2 and 3 of the p-STING blot). (F) Treatment of siRNA against cGAS significantly depletes cGAS and also returns p-STING to WT levels in the VPS13C^KO clones. cGAS knockdown also causes an increase in total STING levels in both WT and VPS13C^KO cells. (G and H) P-TBK1 and p-STING are returned toward WT levels in VPS13C^Rescue clones (G), quantified in H. n = 3 biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. #, P < 0.05; ##, P < 0.01; ###, P < 0.001 in siCGAS or siSTING compared with siScr-treated cells. Data were compared using two-sided t tests. Error bars represent ±SD. Source data associated with this figure can be found at https://doi.org/10.5281/zenodo.6416363. Source data are available for this figure: SourceData F3.