Fig 2: Effects of IL13Rα1 on trail expression in RA FLSs. a RA FLSs (n = 3) were transfected with siIL13Rα1 or siCtrl. Total RNA was extracted and subjected to qRT-PCR for analyzing the changes of trail expression. b The expression of trail at RNA level was detected when RA FLSs (n = 3) was transfected with Lv-IL13Rα1 or Lv-Ctrl. c Phosphorylated and total STAT6 protein were detected in Lv-IL13Rα1 or Lv-Ctrl transfected RA FLSs following Tm or CoCl2 stimulation. d, e RA FLSs (n = 3) harboring Lv-IL13Rα1 or Lv-Ctrl were stimulated with STAT6 inhibitor. Total RNA and protein were subjected to qRT-PCR (d) and Western blot (e) for analyzing trail expression at both RNA and protein levels. With similar treatment in d and e, cell apoptosis (f) and Bax or Bcl protein (g) were detected by Annexin V staining and Western blot. Band intensities are quantified and the Bax/Bcl ratio is summarized into bar charts (right). Data are expressed as the mean of three RA samples ± SD and represent three independent experiments (n = 3). Data in a and b: *p < 0.05 vs the mean ± SD of siCtrl or Lv-Ctrl in the presence of ER stressor. Data in d, f, and g: *p < 0.05 vs the mean ± SD of Lv-IL13Rα1 with vehicle

Fig 3: Mitochondrial STAT6 accumulation leads to tumorigenesis inhibitionIn vivo results on xenograft mouse models.(A–C) Parental or STAT6 KO-293T stable cell line (2 × 106 cells) was injected subcutaneously into the left or right flank of nude mice (n = 18).(A) Representative images of xenograft tumor.(B) Tumor growth curve was measured over time. Data are plotted as mean ± SEM with error bars by two-way repeated measures ANOVA. (∗∗∗∗p < 0.0001) (C) Weight of xenograft tumors. Data represent mean ± SD with error bar (n = 18). ∗∗p < 0.01 (D–H) STAT6 KO-293T, STAT6 WT, ΔSH2, and ΔMTS were inoculated subcutaneously into the left (STAT6 KO) or right (STAT6 WT, ΔSH2, or ΔMTS) flank of nude mice.(D–F) Representative images of xenograft tumors.(G) Tumor growth curve was measured. Data are plotted as mean ± SEM with error bars by two-way repeated measures ANOVA and Tukey’s post-hoc test. ∗∗∗∗p < 0.0001 (STAT6 WT or ΔSH2 vs STAT6 KO).(H) Weight of xenograft tumors. Data represent mean ± S.D. with error bars by one-way ANOVA and Tukey’s post-hoc test. (STAT6 KO, n = 35; STAT6 WT, n = 12; ΔSH2, n = 10; ΔMTS, n = 13). ∗∗p < 0.01 (STAT6 WT or ΔSH2 vs STAT6 KO) and ∗∗∗p < 0.001 (STAT6 WT or ΔSH2 vs ΔMTS).

Fig 4: Co-expression genetic networks associated with Trem2 expression and IL-4-treatment. (A) Co-expression network associated with Trem2 expression, using RNA-seq derived gene expression from primary microglia of Trem2 R47H KI and WT mice. Network contains key genes mediating microglial functions, and includes Trem2, Spi1/PU.1, Tyrobp and Stat6. Larger green spheres represent ‘hub’ genes, those showing the greatest number of connections to other genes in the network, which are likely to play important roles in driving microglial functions alongside Trem2. (B) Co-expression network associated with IL-4-treatment, using RNA-seq derived gene expression from primary microglia treated with IL-4 and untreated. Network includes key genes involved in anti-inflammatory processes, such Arg1, Retnla and Chil3. Larger red spheres represent ‘hub’ genes, which are likely to play important roles in driving the microglial response to IL-4. N = 3 mice per genotype per condition.

Fig 5: Zip9 modulates the macrophage polarity. (A) The siRNA knockdown efficiencies of Zip9. Zip9 was analyzed by RT-PCR following transfection with Zip9 siRNA 48 hours. (B) The analysis of M1-polarized macrophages characteristic genes (Inos, Il6, Il1b, Tnfa). Genes were detected by RT-PCR, following transfection with control small interfering RNA (siRNA) or Zip9 siRNA and stimulated with LPS plus IFNγ. (C) The analysis of M2-polarized macrophages characteristic genes (Ym1, Arg1, Fizz1, Il10, Il4, Tgfb). Genes were detected by RT-PCR, following transfection with control siRNA or Zip9 siRNA and stimulated with IL-4 and IL-13 (20 ng/ml each) *P < 0.05, **P < 0.01 and ns, not significant. Data were analyzed by Student’s t-test. Results were presented as the average ± SD of at least three independent experiments. (D) PEMs were pretreated with control small interfering RNA (siRNA) or Zip9 siRNA for 48h followed by 30/60 mins stimulation with IFNγ and LPS. Cell lysates were processed for immunoblotting with antibody against phospho-IκBα/β and phospho-p65 followed by stripping and reprobing with antibody against α-tubulin. (E) PEMs were pretreated with control small interfering RNA (siRNA) or Zip9 siRNA for 48h followed by 15/30 mins stimulation with IL-4 and IL-13. Cell lysates were processed for immunoblotting with antibody against phospho-STAT6 and total-STAT6 followed by stripping and reprobing with antibody against α-tubulin. Each panel is a representative experiment of at least three independent biological replicates.