Extended Data Fig. 3. SPSB3 targets nuclear cGAS.
a, Results from the siRNA screen highlighting cGAS-GFP nuclear abundance by mean fluorescence intensity and integrated fluorescence intensity in cells treated with siRNAs against SPSB family members (n = 3). b, Nuclear cGAS measurements by confocal microscopy in HeLa cells (endogenous cGAS) or U2OS cells (cGAS-GFP expression) transfected with siRNAs against CUL5, SPSB3 and PSMA5 or control siRNA or treated with epoxomicin or DMSO (n = 2). c, Relative nuclear cGAS-GFP MFI measurement in post-mitotic U2OS cells treated with siRNA against SPSB3 (n = 15) or CUL5 (n = 15), control siRNA (n = 14) or epoxomicin (n = 15). d, Nuclear cGAS-GFP measurements by confocal microscopy in HeLa cells treated with siRNA against SPSB3 (n = 31) or CUL5 (n = 33) or control siRNA (n = 22). e, Whole cell lysates, cytosolic and nuclear fractions were extracted from HeLa cells transfected with non-targeting control siRNA of siRNAs targeting SPSB3 or CUL5 and analysed by immunoblot. Vinculin and H2B were used as loading control of whole cell lysates, cytosolic and nuclear fractions, respectively. f, mRNA levels of cGAS were measured by RT-qPCR in HeLa cells treated with non-targeting control siRNA or siRNAs against SPSB3 or CUL5 for 5 days. Ratios of relative cGAS mRNA levels normalized to the control are shown (n = 3). g, QIBC analysis of endogenous cytosolic cGAS level in HeLa cells treated with siRNA against SPSB3, CUL5 or control siRNA. h, Nuclear cGAS-GFP measurements by confocal microscopy in HeLa cells treated with epoxomicin (n = 32), MLN4924 (n = 29), or DMSO (n = 32). i, QIBC analysis of endogenous cGAS levels in HeLa cells treated with MLN4924 or DMSO. j, mRNA levels of cGAS were measured by RT-qPCR in HeLa cells treated with MLN4924 or DMSO. Ratios of relative cGAS mRNA levels normalized to the control are shown (n = 6). k, Cytosolic and nuclear fractions were extracted from HeLa cells expressing doxycycline (Dox)-inducible SPSB3 that were treated or not with Dox for 4 days). Immunoblots probing cGAS and SPSB3 (FLAG) are shown. Vinculin and H2B were used as loading control for cytosolic and nuclear fractions, respectively. l, Whole cell lysates and nuclear fractions collected from primary endothelial cells, BJ-5ta fibroblasts or differentiated THP-1 cells treated with non-targeting control siRNA or siRNA against SPSB3 for 5 days were analysed by immunoblot. Vinculin and H2B were used as loading control for whole cell lysates and nuclear fractions, respectively. Numbers indicate individual cells (c, d, h) or technical replicates(a, f, j). Data are mean ± SD (a, c, d, f, g, h, j) or mean ± SEM (i). P values were obtained by two-way ANOVA with Tukey’s multiple comparison test (c), one-way ANOVA with Dunnett’s multiple comparison test (d, h), one-way ANOVA (f), one-way ANOVA with Šídák’s multiple comparison test (g) or two-tailed Student’s t-test (j). One representative of three (e, f, k-l) independent experiments is shown.