Fig 2: Inflammatory ligands activate NF-kB and Il6 secretion through an IRE1 pathway in 3T3-L1 adipocytes.A–D, NF-κβ activation was measured in HEK-Blue NOD1 (A, B, D) or TLR4 (C) cells and reported relative to the vehicle control. Cells were treated with vehicle, 10 μg PGN (A), 10 ng/ml TNF (B), 500 ng/ml LPS (C), or 2 μM isoproterenol (D), in the presence or absence of 1 μM KirA6 (N = 12). E, Il6 secretion from differentiated 3T3-L1 adipocytes was measured by ELISA after 48 h of incubation in either the presence or absence of 1 μM KirA6, treated with vehicle, 10 μg/ml PGN, 1 μM thapsigargin, 2 μM isoproterenol, 500 ng/ml LPS, or 10 ng/ml TNF (N = 6). F, Il6 secretion from bone-marrow-derived macrophages was measured by ELISA after 48 h of incubation with or without 1 μM KirA6 and 10 μg/ml PGN, 500 ng/ml LPS, 10 ng/ml TNF, or 1 μM thapsigargin (N = 4). G, glycerol release rates were measured in 3T3-L1 adipocytes treated with or without Il6 (20 ng/ml) in the absence or presence of 1 μM KirA6 (N = 9). H, NF-κβ activation was measured via in HEK-Blue NOD1 cells with or without Il6 (20 ng/ml) in the absence or presence of 1 μM KirA6 (N = 8). Values are mean ± SEM. Statistical significance was measured as p < 0.05 using two-way ANOVA (A–D; G, H). Post hoc analysis was performed using Tukey’s multiple comparisons test. Conditions with different letters (a, b) denote a statistical difference compared with all other conditions without the same letter (A–D). Student t-test was used to determine statistical significance in panels E and F. ∗ denotes significance p < 0.05.

Fig 3: Penfluridol prevents the onset and severity of arthritis in hTNF-TG mice. TNFα overexpression mouse model (hTNF-TG) which can spontaneously develop arthritis were used to observe the inhibitory effect of penfluridol on TNFα-induced NF-κB activation in vivo. Drugs were administrated by oral gavage once a day. Prevention treatment is penfluridol was given to mice at the eighth week of age when arthritis symptom began its onset; treatment of penfluridol was administrated when average clinical arthritis scores reached 8 points in each group. A Clinical score of prevention treatment. B Deformity score of prevention treatment. C Body weight of prevention treatment. D Representative pictures of both front and hind paws in each prevention treatment groups. E H&E staining of paraffin-embedded ankle slides in prevention treatment groups. F Quantitative analysis of inflammation score and pannus formation and cartilage damage score of E. G H&E staining of paraffin-embedded knee sections in preventive treatment groups. H Quantitative analysis of inflammation score and pannus formation and cartilage damage score of G. I Serum IL-1β and IL-6 levels in preventive treatment groups, detected by ELISA. J Clinical scores in treatment groups. K Serum levels IL-1β and IL-6 in treatment groups, detected by ELISA. Six mice in each group. One-way analysis of variance and Bonferroni post hoc test were used to test statistical significance of the differences among groups, and repeated measures analysis of variance was used to test statistical differences of clinical scores among different groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). PF: penfluridol

Fig 4: Activation of JAK1/2 in IFN?R1KO cells is not mediated by extrinsic signals.a mRNA expression of Il6 (*p = 0.0153) and Il6r (*p = 0.0216) in scrambled control (n = 3) and IFN?R1KO (n = 3) cells by real-time RT-PCR. Representative data from two independent experiments are shown as means ± SEM. b, c p-JAK2 in scrambled control and IFN?R1KO cells pretreated with various doses of blocking antibodies against IL-6 (b) or IL-6R (c), analyzed by Western blot (WB). Experiments were repeated twice with similar results. d mRNA expression of Ifnar1 in scrambled control (n = 3) and IFN?R1KO (n = 3) cells by real-time RT-PCR. Representative data from two independent experiments are shown as means ± SEM. ***p = 0.0005. e–g Scrambled and IFN?R1KO cells were transduced with different sgRNAs against mouse Ifnar1. Successfully transduced cells were analyzed for IFNaR1 expression in untreated cells (e) and PD-L1 expression after stimulation with 100 ng/mL IFN-a for 48 h (f) by flow cytometry (FACS strategy 1) and p-JAK1/2 in untreated cells by WB (g). h p-JAK2 in scrambled and IFN?R1KO cells incubated with supernatants (SN) harvested from scrambled or IFN?R1KO cultures for 24 h, analyzed by WB. ß-actin was used as a loading control in WB. All the experiments were repeated twice with similar results. A two-sided Student’s t-test was used for statistical analyses in a, d. Source data are provided in the Source Data file.

Fig 5: Inflammatory signal upregulates Gp2 expression in pancreatic acinar cells.a Cultured pancreatic acinar cells are shown. b Pancreas of control (upper) or Ptf1aCre-ERTMCre-tdTomato (bottom) mice received tamoxifen are shown. c Cultured pancreatic acini from control of Ptf1aCre-ERTM-tdTomato mice stimulated with 4OHT are shown. Data are representing for three independent experiments. d Gp2 expression in no stimulation (None) or stimulated by 200 ng/mL LPS, 20 ng/mL TNF, 20 ng/mL IL-6 (n = 10 /group), 100 ng/mL RANKL (n = 4) pancreatic acini were examined. Relative expressions mRNA levels were determined by quantitative RT-PCR and normalized to Gapdh. *p = 0.040 (one-way ANOVA). Data are presented as mean values ± SEM., N.S. indicates not significant. e Pancreas of mice systemically administered PBS (control) or TNF (100 ng, two times). GP2 (red), DAPI (blue). Representative data of three independent experiments are shown. f GP2 concentration in pancreatic juice. *p = 0.04 (two-tailed Mann–Whitney U test). Data are presented as mean values ± SEM. g TNF concentration in serum *p = 0.021 (two-tailed unpaired t-test). Data are presented as mean values ± SEM. h Representative photograph showing the abdominal-organ matrices surrounding the pancreas in Ptf1aCre-ERTM-tdTomato mice that received tamoxifen. i TNF concentration in abdominal-organ matrices, as determined by enzyme-linked immunosorbent assay. Pancreas (intact n = 15, colitis n = 13), Omentum (intact n = 10, colitis n = 11) mesenteric fat (intact n = 15, colitis n = 13), Retroperitoneal fat and Epididymis fat (intact n = 3, colitis n = 3) *p < 0.05 (two-tailed unpaired t-test). Data are presented as mean values ± SEM. j 100 µg of anti-TNF antibody (Biolegend, MP6-XT22, #506332) or Isotype Rat IgG2a (Biolegend, RTK2758, #400503) was intraperitoneal administered three times (on days 1, 3, and 5) during DSS treatment, and pancreases were collected on day 6. GP2 (red), DAPI (blue). Representative data of three independent experiments are shown. Source data are provided as a Source Data file.