Fig. 3: The NCF1^90H allele upregulates IFN-α/JAK1/STAT1 pathway in mouse macrophages.

a Intracellular ROS in BMDMs without (Blank) and with (Rosup) stimulation (n = 8 per group). Intracellular ROS in BMDMs with NOX2 inhibitor GSK2795039 (n = 5 per group). R90 vs 90H, or m1J. Data were analyzed using one-way ANOVA and presented as mean ± SEM. R90 vs GSK-R90, 90H vs GSK-90H, or m1J vs GSK-m1J, Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. b Extracellular ROS production in BMDMs without (Blank) and with (PMA) stimulation (n = 5 per group). Extracellular ROS in BMDMs after treatment with NOX2 inhibitor GSK2795039 (n = 3 per group). R90 vs GSK-R90, 90H vs GSK-90H, m1J vs GSK-m1J. R90 vs 90H or m1J, 90H vs Hank’s Balanced Salt Solution (HBSS). Data were analyzed using one-way ANOVA and presented as mean ± SEM. R90 vs GSK-R90, 90H vs GSK-90H, or m1J vs GSK-m1J, Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. c Western blots of JAK1, STAT1, and STAT3 proteins in naïve macrophages, and after IFN-α stimulation from 90H and m1J male mice, compared to wild-type (R90) male mice (n = 5 per group). d, e Quantification of relative protein expression, normalized to β-actin (n = 4 per group). Quantitative comparisons between samples on the same blots or the samples derive from the same experiment and that blots were processed in parallel. R90 vs 90H, R90 vs m1J. IFN-α R90 vs IFN-α 90H, IFN-α R90 vs IFN-α m1J. Data were analyzed using one-way ANOVA and presented as mean ± SEM. f Western blots of p-JAK1, p-STAT1, and p-STAT3 in IFN-α stimulated macrophages, together with adding Ncf1/NOX2 blocker to cells from wild-type mice (n = 5 per group), or adding H₂O₂ to cells from 90H or m1J mice (n = 5 per group). g–i Quantification of relative protein expression, normalized to β-actin (n = 5 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. j Representative histograms of STAT1 and p-STAT1 in peritoneal exudates macrophages (CD45⁺ CD11b⁺ F4/80⁺ Ly6C⁻) one- day post-MNV injection (1dpi) by IP. k Geometric mean of STAT1 and p-STAT1, and relative expression of p-STAT1/STAT1 within peritoneal exudates macrophages (MNV-R90: female: n = 4, male: n = 4; MNV-90H: female: n = 5, male: n = 4). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. BMDMs were obtained from the differentiation of monocytes recovered from the femur and tibia of 6 to 8- week- old male B6N.Q.Ncf1^R90, B6N.Q. Ncf1^90H, B6N.Q and B6N.Q.Ncf1^m1J mice.