Fig 1: Characteristics of IFN signaling induction in ATAD3A patient–derived fibroblasts. (A) ATAD3A mRNA expression in primary fibroblasts from controls (HDF; average of three) or from ATAD3A patients 2, 3, and 4 (AGS1530, AGS2216, and AGS2792, respectively). Mean values and data points of 10–14 independent experiments are shown and are expressed as the ratio of the mRNA levels, normalized to housekeeping gene HPRT mRNA, in indicated conditions to that in one control HDF. * indicates statistical significance (P < 0.05) in one-way ANOVA with Dunn’s multiple comparison test. (B) Protein expression of ATAD3A in primary fibroblasts assessed by Western blot. VDAC1 is a mitochondrial protein, and vinculin a loading control. (C) qPCR analysis of mtDNA copy number per cell, expressed as the ratio of the DNA quantity of the mitochondrial gene (MT-COXII) over the nuclear gene (GAPDH), in total DNA isolated from fibroblasts. The mean of eight independent experiments is shown. * indicates statistical significance (P < 0.05) in one-way ANOVA with Dunn’s multiple comparison test. (D) Representative ISG (Mx1, RSAD2, OAS1, and IFI27), cGAS, and MAVS mRNA expression, expressed as in A, in control and ATAD3A patient–derived primary fibroblasts, after down-regulation of cGAS (sicGAS) or MAVS (siMAVS) by siRNA or treatment with control siRNA (siCTRL). Mean values and data points of six to nine independent experiments are shown. * indicates statistical significance (P < 0.05) in two-way ANOVA with Dunnett’s multiple comparison tests between siRNA conditions for each cell line. # indicates statistical significance (P < 0.05) in two-way ANOVA with Dunnett’s multiple comparison test between control and patient-derived fibroblasts for each siRNA condition. (E) Representative ISG (IFIT1 and Mx1) and IFNb mRNA expression, expressed as in A, in control (HDF; three lines pooled) and ATAD3A patient–derived primary fibroblasts (Pt; three lines pooled), UT) or mtDNA-depleted by ddC as in Fig. 4 D, and stimulated with 1 µg/ml HT-DNA or lipofectamine (Lipo) alone for 24 h. Mean values and data points of three independent experiments are shown. ns indicates nonsignificance in two-way ANOVA with Sidak’s multiple comparison test between UT and ddC conditions. (F) Representative ISG (Mx1, OAS1, ISG15, and IFI27), ATAD3A, and TFAM mRNA expression, expressed as in A, in control and ATAD3A patient–derived primary fibroblasts, UT or mtDNA depleted by ddC as in Fig. 4 D. Mean values and data points of three to eight independent experiments are shown. * indicates statistical significance (P < 0.05) in two-way ANOVA with Sidak’s multiple comparison test between UT and ddC conditions. # indicates statistical significance (P < 0.05) in two-way ANOVA with Dunnett’s multiple comparison test between control and patient-derived fibroblasts for each treatment. (G) Representative ISG Mx1 and ATAD3A mRNA expression in fibroblasts vehicle-treated (DMSO) or treated with 300 µM DIDS or 100 nM rapamycin for 72 h. Mean values and data points of five to seven independent experiments are shown. * indicates statistical significance (P < 0.05) in two-way ANOVA with Sidak’s multiple comparison test between treatment conditions. # indicates statistical significance (P < 0.05) in two-way ANOVA with Dunnett’s multiple comparison test between control and patient-derived fibroblasts for each treatment. (H) ISG mRNA expression, represented as an ISG score, and BAK1 mRNA expression expressed as in A, in fibroblasts after down-regulation of BAX (siBAX) or BAK1 (siBAK1) or both (siBAX+siBAK1) by siRNA or treatment with control siRNA (siCTRL). Mean values and data points of two to three independent experiments are shown. * indicates statistical significance (P < 0.05) in two-way ANOVA with Dunnett’s multiple comparison tests between siRNA conditions for each cell line. # indicates statistical significance (P < 0.05) in two-way ANOVA with Dunnett’s multiple comparison test between control and patient-derived fibroblasts for each siRNA condition. (I) Protein BAK1, BAX, and ATAD3A levels in primary fibroblasts assessed by Western blot; VDAC1 is a mitochondrial protein, and vinculin and cofilin are loading controls. (J) ISG mRNA expression, represented as an ISG score, in fibroblasts after treatment with 50–200 µM of BAX inhibitor peptide V5 (BAXi V5) for 72 h. Mean values and data points of three to four independent experiments are shown. # indicates statistical significance (P < 0.05) in two-way ANOVA with Dunnett’s multiple comparison test between control and patient-derived fibroblasts for each treatment.