Fig 2: STAT1-mediated control of PARP1 levels in cervical cancer models.a Relative mRNA expression level of STAT1, STAT3, PSME1, PSME2, and PARP1 was evaluated by RT-qPCR in CaSki, C-4I, HeLa S3, and C33A cells. Samples were normalized to the mean of two housekeeping genes, GAPDH and PGK1. For each mRNA, results are presented as fold change compared to CaSki cells (mean ± SD, n = 3). Statistical significances have been evaluated through an unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001 respect to CaSki cells. b Representative Western blot analysis of STAT1, STAT3, PA28a, PA28ß, PARP1 proteins in CaSki, C-4I, HeLa S3, and C33A cells. GAPDH was used as a control. c Representative pictures showing immunolocalization of PARP1 in CaSki and C-4I cells (magnification 63x). d, e Representative Western blot of STAT1, STAT3, PA28a, PA28ß, PARP1 proteins in CaSki, and C-4I cells. Cells were harvested 48 h following STAT1 silencing (d) or 10 ng/ml of IFN-? treatment (e) and whole-cell lysates (10 µg) were loaded into SDS-PAGE, followed by Western blot with specific antibodies. ß-Actin was used as a loading control. f CaSki and C-4I cells were treated with 10 ng/ml IFN-? for 24 h and then added MG-132 (0.5 µM) for an additional 24 h. Cells were harvested and whole-cell lysates (10 µg) were loaded into SDS-PAGE, followed by Western blot with specific antibodies. ß-Actin was used as a loading control. siC siRNA control, siSTAT1 siRNA targeted to STAT1, CTR untreated, IFN-? interferon-?. Data were representative of at least three experiments.

Fig 3: Macrophage NCOR1 (nuclear receptor corepressor 1) deficiency suppresses interleukin (IL)-1ß- or IL-6-induced inflammation in macrophages. Peritoneal macrophages were isolated from littermate control and macrophage Ncor1 knockout mice. A, Quantitative reverse transcription polymerase chain reaction analysis of inflammatory genes in macrophages stimulated with IL-1ß for 6 hours. B, Western blotting analysis of phosphorylation and total protein levels of STAT1, P38, P65, and IKKß in peritoneal macrophages treated with IL-1ß for indicated time periods. C, Quantitative reverse transcription polymerase chain reaction analysis of inflammatory genes in macrophages stimulated with IL-6 for 6 hours. D, Western blotting analysis of phosphorylation and total protein levels of STAT1, P38, P65, and IKKß in peritoneal macrophages treated with interleukin-6 for indicated time periods. Representative results of quantitative reverse transcription polymerase chain reaction and western blotting from 3 independent experiments were shown. Values are expressed as mean±SEM. Cox2 indicates prostaglandin-endoperoxide synthase 2; Cxcl1, C-X-C motif chemokine ligand 1; IKKß, inhibitor of kappaB kinase beta; Il-1b or IL-1ß, interleukin 1 beta; Il-6 or IL-6, interleukin 6; LC, littermate control; MNKO or KO, macrophage Ncor1 knockout; Nos2, nitric oxide synthase 2; ns, not significant; STAT1, signal transducer and activator of transcription 1; and Tnfa, tumor necrosis factor. *P<0.05, **P<0.01, ***P<0.001.

Fig 4: Neutralization of IFNγ reduces Con A-induced Stat1 activation and liver damage. (A) Con A (10 mg/kg) was intravenously injected into WT and Spred2-/- mice. The level of each protein in liver extracts at 12 h post Con A challenge was evaluated by Western blotting using the indicated primary antibodies (3 mice, each). Band densities were semi-quantitated at 12 h post Con A challenge. (B) Spred2-/- mice were intraperitoneally administered with anti-mouse IFNγ IgG (1 mg/mouse) or control rabbit IgG 12 h prior to 10 mg/kg Con A challenge. The level of each protein in liver extracts at 12 h post Con A challenge was evaluated by Western blotting using the indicated primary antibodies (3 mice, each). Band densities were semi-quantitated. (C) Spred2-/- mice were intraperitoneal injected with 10 μmol/kg U0126 or DMSO 3 h prior to 10 mg/kg Con A challenge. The level of each protein in liver extracts at 12 h post Con A challenge was evaluated by Western blotting using the indicated primary antibodies (3 mice per group). Band densities were semi-quantitated. (D, E) Anti-mouse IFNγ IgG (1 mg/mouse) or control rabbit IgG was intraperitoneally injected into Spred2-/- mice 12 h prior to 10 mg/kg Con A challenge. (D) Serum ALT levels at 12 h post Con A challenge were measured (control; 4 mice, anti-IFNγ-treated; 6 mice). (E) Left; representative photographs of H&E-stained liver sections (scale bar, 100 µm) are shown. Right; %injured area was estimated using the cellSens Standard software (control; 4 mice, anti-IFNγ-treated; 6 mice). *p < 0.05., **p < 0.01.

Fig 5: Inflammation and activation of the STAT1/NOS2 pathway in HI skin.Representative (A) IL-37, (C) IL-36a, (E) IL-36?, (G) STAT1, (I) p-STAT1, (J) and NOS2 (green channel) and DAPI (blue channel) staining images of control skin and HI patient skin. Arrows indicate granular layer. Associated quantitative analysis of (B) IL-37, (D) IL-36a, (F) IL-36?, (H) granular layer STAT1, and (K) NOS2 protein expression in control skin and HI patient skin. Each dot represents the mean of relative protein intensity from 3 independent images. n = 3 or 4. Data are represented as mean ± SD. *P = 0.05; **P = 0.01; ***P = 0.001, unpaired t test. Scale bars: 100 µm.