Figure S2.
XIST RNA localization profiles and impact of IFN-I signaling on XCI escape and expression levels of TLR7 in female pDCs. (A) RNA FISH representative images of XIST patterns I–IV from HD (upper panels) and SSc donors (lower panels). Arrowheads point to the different XIST patterns. Scale bars 1 µm. (B) Frequencies of XIST patterns, comparing HDs versus SSc pooled patients in four independent experiments (P#01 to P#04). (C and D) Graphs representing the frequencies among (C) the I and II XIST patterns, and (D) the frequencies of TLR7 biallelic nuclei among pDCs with Type-III and -IV XIST patterns in paired experiments (n = 4). Statistical differences were analyzed using a paired Student’s t test. (E) mRNA TLR7 positive cells in 96-wells plate from HDs (n = 5) and SSc donors (n = 5). Statistical analysis was performed using a Mann–Whitney test. (F) Frequencies of TLR7 biallelic cells, relative to the total TLR7+ nuclei in IL-3-pDCs stimulated or not with IFN-β for 2 h before RNA-FISH (n = 4, pool#5 to #8). (G) Frequencies of TLR7 biallelic nuclei among total nuclei counted from all the pooled donors IFNβ− (n = 430) and IFNβ+ (n = 713). Statistical differences were analyzed using a Fisher’s exact test. (H) Frequencies of XIST patterns I–IV comparing cells incubated in medium (−IFN-β) or with IFN-β (n = 4, pool#5 to #8). Statistical analysis performed with a repeated measured one-way ANOVA test. (I) Frequencies of nuclei with XIST Type-I and -II pattern calculated from the total nuclei from all pooled donors, with Medium (IFNβ−; n = 1,424) and IFNβ+ (n = 1,810). Statistical analysis was performed using a Fisher exact test. (J) mRNA was isolated from 41 PBMCs samples incubated or not with IFN-β (1 ng/ml) for 3 h. TLR7 and MxA gene expression was analyzed by RT-qPCR and normalized over GAPDH expression. (K) mRNA was isolated from purified pDCs incubated or not with IFN-β (1 ng/ml) for 3 h. TLR7 and MxA gene expression was analyzed by RT-qPCR and normalized over GAPDH expression. Statistical analyses were performed with a Wilcoxon test (J) and a Student’s paired t test (K). *P < 0.05; **P < 0.01; ***P < 0.001.

XIST RNA localization profiles and impact of IFN-I signaling on XCI escape and expression levels of TLR7 in female pDCs. (A) RNA FISH representative images of XIST patterns I–IV from HD (upper panels) and SSc donors (lower panels). Arrowheads point to the different XIST patterns. Scale bars 1 µm. (B) Frequencies of XIST patterns, comparing HDs versus SSc pooled patients in four independent experiments (P#01 to P#04). (C and D) Graphs representing the frequencies among (C) the I and II XIST patterns, and (D) the frequencies of TLR7 biallelic nuclei among pDCs with Type-III and -IV XIST patterns in paired experiments (n = 4). Statistical differences were analyzed using a paired Student’s t test. (E) mRNA TLR7 positive cells in 96-wells plate from HDs (n = 5) and SSc donors (n = 5). Statistical analysis was performed using a Mann–Whitney test. (F) Frequencies of TLR7 biallelic cells, relative to the total TLR7+ nuclei in IL-3-pDCs stimulated or not with IFN-β for 2 h before RNA-FISH (n = 4, pool#5 to #8). (G) Frequencies of TLR7 biallelic nuclei among total nuclei counted from all the pooled donors IFNβ− (n = 430) and IFNβ+ (n = 713). Statistical differences were analyzed using a Fisher’s exact test. (H) Frequencies of XIST patterns I–IV comparing cells incubated in medium (−IFN-β) or with IFN-β (n = 4, pool#5 to #8). Statistical analysis performed with a repeated measured one-way ANOVA test. (I) Frequencies of nuclei with XIST Type-I and -II pattern calculated from the total nuclei from all pooled donors, with Medium (IFNβ−; n = 1,424) and IFNβ+ (n = 1,810). Statistical analysis was performed using a Fisher exact test. (J) mRNA was isolated from 41 PBMCs samples incubated or not with IFN-β (1 ng/ml) for 3 h. TLR7 and MxA gene expression was analyzed by RT-qPCR and normalized over GAPDH expression. (K) mRNA was isolated from purified pDCs incubated or not with IFN-β (1 ng/ml) for 3 h. TLR7 and MxA gene expression was analyzed by RT-qPCR and normalized over GAPDH expression. Statistical analyses were performed with a Wilcoxon test (J) and a Student’s paired t test (K). *P < 0.05; **P < 0.01; ***P < 0.001.

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