Silencing of cGAS unmasks cGAMP responsiveness and reveals impaired STING degradation in VPS13C ^KO cells. (A–C) Treatment with siRNA against cGAS returns STING-GFP to an ER distribution in VPS13C^KO cells, similar to untreated WT cells (A), quantified in B, while siScr has no effect, quantified in C. n = 3 biological replicates. (D) IB against STING in WT and VPS13C^KO cells treated with either scrambled siRNA or siRNA against CGAS, followed by treatment with 8 µg/ml cGAMP for 24 h. Treatment with siRNA against CGAS returns STING to the unphosphorylated state in VPS13C^KO cells, rendering them responsive to cGAMP. In VPS13C^KO cells, the activation of STING is not followed by degradation, compared with WT cells. (E) Quantification of the total STING signal remaining 24 h after cGAMP treatment (F₂₄) relative to 0 h (F₀). For quantification of the STING bands, both the upper and lower band were included. All bands were normalized to the loading control. n = 3 biological replicates. (F) IB against p-TBK1 under the same conditions as E. WT cells behaved similarly in response to cGAMP treatment regardless of siScr or siCGAS pretreatment, with p-TBK1 levels returning to baseline after 24 h of cGAMP treatment, as shown by the time course of Fig. 5, F and G. In VPS13C^KO cells pretreated with siScr, p-TBK1 remained at baseline after 24 h of cGAMP treatment, presumably never having increased, based on the time course in Fig. 5, F and G. In VPS13C^KO cells pretreated with siCGAS, however, p-TBK1 was significantly elevated after 24 h of cGAMP, in accordance with the defect in STING degradation and continued STING signaling (E and F). (G) Quantification of p-TBK1 band intensity. All bands were normalized to the loading control. n = 3 biological replicates. Scale bars, 20 μM. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 compared with untreated WT cells. ##, P < 0.01; ###, P < 0.001 value at 24-h cGAMP treatment compared with corresponding 0-h cGAMP treatment. 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 F6.