Fig 1: PARP1 activates JNK pathway via PARP1‐JNK interaction in an oxidate stress‐induced inflammatory environment. A) The subcellular distribution of PARP1 and JNK protein in NCM460 and Caco2 cell lines was detected by confocal microscopy. Scale bars: 10 µm. B,C) Anti‐PARP1 B) and anti‐JNK C) antibodies were respectively applied in the Co‐IP of STC1 OE NCM460 cells. D) His pull‐down assays of purified recombinant human His‐PARP1 and JNK protein. E) After the application of PARP inhibitor PJ34 (5µM, 24 h), the expression of JNK pathway protein in NCM460 and Caco2 cells was detected via western blot. F) The expression level of JNK pathway protein in the colonic tissues of WT, Stc1 INT‐KO, WT+DSS, and Stc1 INT‐KO+DSS mice was detected via western blot. G) The expression level of JNK pathway protein in H2O2‐treated STC1 OE and STC1 KO cells were detected via western blot.
Fig 2: Stc1 deficiency alleviates DSS‐induced acute murine colitis. A) Body weight loss of WT, Stc1 INT‐KO, WT+DSS, and Stc1 INT‐KO+DSS mice. WT, n = 6; Stc1 INT‐KO, n = 7; WT+DSS, n = 8; Stc1 INT‐KO+DSS, n = 8. (B, C) Mice colon length and representative images of colons. WT, n = 6; Stc1 INT‐KO, n = 7; WT+DSS, n = 8; Stc1 INT‐KO+DSS, n = 8. D) DAI scores were used in the evaluation of colitis severity. WT, n = 6; Stc1 INT‐KO, n = 7; WT+DSS, n = 8; Stc1 INT‐KO+DSS, n = 8. (E, F) Histological scores and representative colonic histopathological images. WT, n = 6; Stc1 INT‐KO, n = 7; WT+DSS, n = 8; Stc1 INT‐KO+DSS, n = 8. Scale bars: 200 µm. (G, H) Representative colonoscopy images and endoscopic colitis scores were used to evaluate colitis severity observed under murine colonoscopy. WT, n = 3; Stc1 INT‐KO, n = 3; WT+DSS, n = 6; Stc1 INT‐KO+DSS, n = 6. I) TEM detected the deformation of mitochondria (marked by red arrows) in mice colonic epithelial cells. Scale bars: 1 µm. (J, K) Representative IF images of γ‐H2AX‐ J) and PAR‐stained K) mice colon sections. Scale bars: 100 µm. (L) The expression level of pro‐inflammatory cytokine mRNA in mice colonic tissues was detected via qPCR. WT, n = 5; Stc1 INT‐KO, n = 5; WT+DSS, n = 5; Stc1 INT‐KO+DSS, n = 5. M,N) The expression level of IL‐6 and TNF‐α protein in mice serum was detected via multiELISA. WT, n = 4; Stc1 INT‐KO, n = 4; WT+DSS, n = 4; Stc1 INT‐KO+DSS, n = 4. Data were expressed as mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001.
Fig 3: STC1 interacts with and upregulates PARP1. A) Coomassie blue was used in the preliminary identification of STC1 and target protein from the IP elution of STC1 OE NCM460 cells. B) Potential target proteins based on proteomic analysis. C) The subcellular distribution of STC1 and PARP1 protein in NCM460 and Caco2 cell lines was detected by confocal microscopy. Scale bars: 10 µm. (D, E) Anti‐STC1 D) and anti‐PARP1 E) antibodies were respectively applied in the Co‐IP of STC1 OE NCM460 cells. F) His pull‐down assays of purified recombinant human His‐PARP1 and STC1 protein. G) Protein truncated tests were designed to confirm the interaction between STC1 protein and PARP1 mutants in 293T cells. H) After H2O2‐pretreatment, the degradation of PARP1 protein in Control, STC1 OE, WT and STC1 KO NCM460 and Caco2 cells was detected via western blot, when protein synthesis was inhibited by 50µg mL−1 CHX in indicated time course. I) The ubiquitination of PARP1 protein was detected in the presence or absence of STC1 protein in 293T cells.
Fig 4: STC1 increases in the colonic mucosa of Crohn's disease (CD) patients and in chemically‐induced murine colitis. A) Heatmap of transcriptome sequencing showed STC1 mRNA increased in the inflamed colonic mucosa of CD patients (n = 10) compared with uninflamed colonic mucosa of healthy controls (n = 10). B) The expression level of STC1 mRNA in the inflamed colonic mucosa of CD patients (CD, n = 41) and uninflamed mucosa of healthy controls (NC, n = 25) was examined via qPCR. C) The expression level of STC1 protein in CD and NC colonic mucosa was detected by western blot. D) Representative IHC images of STC1‐stained colon sections from NC and different stages of CD. Scale bars: 200 µm (upper row) and 50 µm (lower row). E) Statistical analysis on the staining area of STC1 in NC and different stages of CD. NC, n = 7; CDAI<150, n = 9; 150≤CDAI<220, n = 9; 220≤CDAI<450, n = 9; CDAI≥450, n = 6. Positive staining area was quantified by ImageJ. F,G) The expression level of Stc1 mRNA in DSS‐ (NC, n = 5; DSS, n = 8) and TNBS‐induced (NC, n = 6; TNBS, n = 6) mice colitis models were examined via qPCR. H,I) The expression level of STC1 protein in DSS‐ and TNBS‐induced colitis models was examined via western blot. J,K) Representative IHC images of STC1‐stained colon sections from DSS‐ and TNBS‐induced colitis models. Scale bars: 50 µm. Data were expressed as mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001.
Fig 5: STC1 intensifies parthanatos and aggravates inflammation in vitro. A,B) KEGG A) and GO B) analysis on the enriched proteins in STC1 OE NCM460 cells. (C, D) Dye eFluor 660 marked dead cells after H2O2‐stimulation in Control, STC1 OE, WT, and STC1 KO NCM460 cells and was detected by flow cytometry. E) The expression level of pro‐inflammatory cytokine mRNA in H2O2‐treated NCM460 cells was detected via qPCR. F,G) Representative IF images and quantification of γ‐H2AX‐stained H2O2‐treated NCM460 cells. Scale bars: 100 µm. H) The expression level of PAR in H2O2‐treated NCM460 cells was detected via western blot. Scale bars: 100 µm. I,J) The nuclear translocation of AIF was detected via western blot I) and IF J). Scale bars: 10 µm. Data were expressed as mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001.
Supplier Page from Abcam for Recombinant Human Stanniocalcin 1/STC protein