Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: The levels of regenerating islet-derived protein 3-alpha (REG3A) was increased and miR-146a was decreased in polymyositis and dermatomyositis (PM/DM) patients. (A) Peripheral blood mononuclear cells (PBMCs) were isolated from patients ( n = 25) and the healthy controls ( n = 20) and the messenger RNA (mRNA) levels of interferon gamma (IFN-γ), interleukin (IL)-17A, REG3A, and miRNA-146a were determined by real-time PCR. The relative mRNA expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/U6. (B) The levels of IFN-γ and IL-17A in serum from patients and the healthy controls were determined by ELISA assay. (C) The REG3A levels from patients and the healthy controls were determined by immunohistochemistry. Data are shown as means ± SEM. * p < 0.05, ** p < 0.01 in comparison with the healthy controls. PBMCs were obtained from 20 healthy controls and 25 patients with PM/DM.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Comparison

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: The levels of regenerating islet-derived protein 3-alpha (REG3A) and miR-146a in an experimental autoimmune myositis (EAM) model. (A) The messenger RNA (mRNA) expression of interferon gamma (IFN-γ), interleukin (IL)-17A, REG3A, and miRNA-146a in muscle tissues were determined by real-time PCR. The relative mRNA expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/U6. ( n = 9) (B) . The levels of IFN-γFand IL-17A in serum from the EAM mice were determined by ELISA assay ( n = 9). (C) The protein levels of REG3A in muscle tissues were determined by Western blot ( n = 3). GAPDH was used as the internal controls for Western blot analysis. Bands were quantified using ImageJ. Data are shown as means ± SEM. * p < 0.05, ** p < 0.01 in comparison with the control group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: The effects of miR-146a and regenerating islet-derived protein 3-alpha (REG3A) on macrophage migration. (A) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were transfected with the negative control (NC) microRNA (miRNA) and miR-146a mimics, miR-146a inhibitors for 24 h. (B) Monocyte-derived macrophages from the peripheral blood mononuclear cells (PBMCs) of the healthy donors ( n = 3) were transfected with the NC siRNA, REG3A siRNA, pcDNA3.1-NC, and pcDNA3.1-REG3A plasmids for 24 h. All treated cells (2 × 10 5 ) were suspended and added to the upper chamber of transwell. The medium containing 10% human serum was used as a chemoattractant in the lower chamber. After incubation for 24 h, the invaded cells into the lower chamber were stained with crystal violet. The migrated cells were counted, and photomicrographs were taken under an Olympus inverted microscope (IX71, Olympus, Japan). Data are shown as means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 in comparison with the control group. ## p < 0.05 in comparison with pcDNA3.1-NC group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Migration, Transfection, Negative Control, Incubation, Staining, Inverted Microscopy, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: Interleukin (IL)-17A induced regenerating islet-derived protein 3-alpha (REG3A) expression in macrophage. (A) Monocyte-derived macrophages from the peripheral blood mononuclear cells (PBMCs) of the healthy donors ( n = 3) were treated with the different concentrations of IL-17A for 24 h. The messenger RNA (mRNA) expression of REG3A was determined by real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal controls. (B) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were treated with the different concentrations of IL-17A for 24 h. The mRNA expression of miR-146a was determined by real-time PCR. U6 was used as the internal controls. (C) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were treated with the different concentrations of IL-17A for 24 h. The protein levels of REG3A were determined by Western blot. GAPDH was used as the internal controls. (D) Monocyte-derived macrophages were transfected with the NC small-interfering RNA (siRNA) and IL-17RA siRNA in the absence or presence of IL-17A for 24 h. The protein levels of REG3A were determined by Western blot. GAPDH was used as the internal controls. Western blot bands were quantified using ImageJ and normalized to GAPDH. Data are shown as means ± SEM of three independent experiment. * p < 0.05, ** p < 0.01 in comparison with the control group. # p < 0.05 in comparison with IL-17A + NC siRNA group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Small Interfering RNA, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: miR-146a regulates regenerating islet-derived protein 3-alpha (REG3A) expression in macrophage. (A,B) Monocyte-derived macrophages from the peripheral blood mononuclear cells (PBMCs) of the healthy donors ( n = 3) were transfected with negative control (NC) microRNA (miRNA), miR-146a mimics, or miR-146a inhibitor for 24 h. The messenger RNA (mRNA) and protein levels of REG3A were determined by real-time PCR and Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal controls. (C) The cells were transfected with NC small-interfering RNA (siRNA), REG3A siRNA, pcDNA3.1-NC, and pcDNA3.1-REG3A plasmids for 24 h. The mRNA levels of miR-146a were determined by real-time PCR. U6 was used as the internal controls. (D,E) The cells were transfected with NC miRNA or miR-146a mimics in the absence or presence of IL-17A for 24 h. The mRNA and protein levels of REG3A were determined by real-time PCR and Western blot. GAPDH was used as the internal controls. Data are shown as means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 in comparison with the control group. # p < 0.05, ## p < 0.01 in comparison with IL-17A treatment group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: miR-146a inhibited macrophage migration through suppression of regenerating islet-derived protein 3-alpha (REG3A) expression. (A) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were transfected with the NC siRNA or REG3A siRNA in the absence or presence of miR-146a inhibitor for 24 h. (B) Monocyte-derived macrophages were transfected with the pcDNA3.1-NC or pcDNA3.1-REG3A plasmids in the absence or presence of miR-146a mimics for 24 h. All treated cells (2 × 10 5 ) were suspended and added to the upper chamber of transwell well. The medium containing 10% human serum was used as a chemoattractant in the lower chamber. After incubation for 24 h, the invaded cells into the lower chamber were stained with crystal violet. The migrated cells were counted, and photomicrographs were taken under an Olympus inverted microscope (IX71, Olympus, Japan). Data are shown as means ± SEM of three independent experiment. * p < 0.05, ** p < 0.01 in comparison with NC + NC siRNA or NC + pcDNA3.1 group. # p < 0.05, ## p < 0.01 in comparison with inhibitor + NC siRNA or NC + pcDNA3.1-REG3A group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Migration, Derivative Assay, Expressing, Transfection, Incubation, Staining, Inverted Microscopy, Comparison

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A) Schematic of stool extract preparation and analysis. B) Western blots of REG1A, REG3A, REG3G, and REG4 in stool extracts from representative 3 non-IBD (NIBD) and 5 IBD patients. C) Proportion of stool specimens from individual NIBD (n = 23) and IBD (n = 26) patients in which REG1A, REG3A, REG3G, or REG4 were detectable by western blot. D) Quantification of REG3A in NIBD and IBD patients-derived stool extracts by ELISA. E) Fold changes in colony forming units (CFU) of Enterococcus faecalis ( Efl ) and Salmonella Typhimurium ( S Tm) following 24 hrs of culture in stool extracts from NIBD (n = 56) and IBD (n = 56) patients. Results obtained with Efl were confirmed with E. faecium ( Efm ) (n = 18 for NIBD and n = 19 for IBD). F) Correlation between REG3A concentration and CFU fold changes of Efl (upper), S Tm (middle), and Efm (lower) cultured in stool extract from NIBD and IBD patients. G) Fold change in Efm CFU at 24 hr-post culture in NIBD (left) and IBD (right) patient-derived stool extract or PBS in the presence of anti-REG1A, REG3A, and REG3G antibodies. N = 13 for NIBD and IBD. H) Correlation between REG3A concentration and Crohn’s disease activity index (CDAI) for CD patients (n = 31, left) or total Mayo score for ulcerative colitis (UC) patients (n = 24, right). Data points in D-H represent individual patients. Bars represent mean ± SEM and at least three independent experiments were performed. NIBD, non-IBD; Efl , E. faecalis ; S Tm, S. Typhimurium; Efm , E. faecium ; CDAI, Crohn’s disease activity index; UC, ulcerative colitis; r, Pearson correlation coefficient. Indicated p values by Fisher’s exact test in C, unpaired t test, two-tailed in D, E, and G, and simple linear regression analysis in F and H.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A) REG3A concentration in stool extracts from NIBD and IBD patients from separated by sex. B) REG3A concentration in stool extracts from Crohn’s disease (CD) or ulcerative colitis (UC) patients. C) Correlation between age of NIBD and IBD patients and REG3A concentration. D) REG3A concentration in stool extracts segregated into indicated age groups. E) Proportion of patients in which Enterococcus and E. faecium ( Efm ) were detected in stool. F) Proportion of Enterococcus that are Efm in NIBD and IBD stools. Data points in A-D and F represent individual patients. Bars represent mean ± SEM and at least two independent experiments were performed. r, Pearson correlation coefficient. Indicated p values by unpaired t test, two-tailed in A, D, and F, simple linear regression analysis in C, and Fisher’s exact test in E.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A and B) 16S rRNA sequencing of stool from NIBD and IBD patients from . Alpha diversity values calculated as Shannon (right), Faith’s phylogenetic diversity (PD) (middle), and Pielou-evenness (right) indices (A). Principle coordinate analyses of beta diversity determined by Bay-Curtis, Jaccard, and Unweighted and Weighted unifrac methods (B). C) Proportion of sequencing reads representing Enterococcus in NIBD and IBD patient stool. One NIBD sample contained >80% Enterococcus indicative of an overabundance, which is shown as a reference on the graph but excluded from statistical analysis and downstream assays. D) Total Enterococcus (left) and Efm (right) CFUs in stool from NIBD (n = 39) and IBD (n = 43) patients. E) Correlation between REG3A concentration and the burden of total Enterococcus and Efm in IBD stool. F) Correlation between the burden of total Enterococcus and CDAI of CD patients (n = 24, left) and total Mayo score of UC patients (n = 18, right). Data points represent individual patients. Bars represent mean ± SEM and at least three independent experiments were performed. r, Pearson correlation coefficient. Indicated p values by Kruskal-Wallis test in A, unpaired t test, two-tailed in C and D, and simple linear regression analysis in E and F.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Sequencing, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A) NOD2 R702W allele frequency according to ethnic groups. The frequencies were retrieved from 1000 Genomes Project and Allele Frequency Aggregator. B) Schematic of genotyping PCR product (left) and representative genotyping gel image (right) for Nod2 Q675W knock-in mouse. C) Sequencing of the Nod2 locus from the above mice confirmed successful gene targeting. Figure shows exon 4 DNA and amino acid sequences from Nod2 Q675W knock-in mouse sequencing results aligned to the WT sequences. Red boxes indicate the mutated region. D) Western blot image of NOD2 and β-actin (ACTB) in colonic tissue lysates from Nod2 -/- , Nod2 Q675W/+ , and Nod2 Q675W/Q675W mice. E and F) DSS treatment of Nod2 Q675W/+ and Nod2 Q675W/Q675W mice from room 6 following 2-week administration of Efm in drinking water or control. The mice were examined for the burden of Efm (E) and LCN2 concentration (F) in the stool samples at the indicated time points. G) Endogenous Enterococcus burden among mice with different genotypes on day 0 (upper). The p -values relative to Nod2 Q675W/+ or Nod2 Q675W/Q675W mice were indicated in lower table. H) Schematic of the mechanism by which Efm activates NOD2 to suppress inflammation, and how this process is disrupted when either REG3A is overproduced or NOD2 is genetically inactivated. Lines in E and data points in F and G represent individual mice. Bars in F and G represent mean ± SEM and at least three independent experiments were performed. Het, heterozygotes; Homo, homozygotes. Indicated p values by unpaired t test, two-tailed in F and G.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Knock-In, Sequencing, Western Blot, Control, Concentration Assay, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Higher peritumoral REG3A expression indicates malignant progression and poor prognosis in patients with PDAC. A The serum concentration of REG3A in healthy volunteers and patients with different TNM stages of PDAC. B The median value was used as the low and high cut-offs to define the REG3A-Low ( n = 28) and REG3A-High ( n = 32) groups. Kaplan–Meier survival analysis of patients with different serum REG3A levels. C Representative images of REG3A expression in a tissue microarray (TMA) cohort of PDAC specimens. D The integral optical density (IOD) analysis of in situ REG3A expression from the TMA PDAC specimens with 54 paired (peritumor vs tumor) samples. E Kaplan–Meier survival analysis of patients with different peritumoral REG3A expression levels (REG3A-Low, n = 32; REG3A-High, n = 22) in the TMA cohort. F The expression of REG3A in tumor tissues compared to peritumoral tissues in patients with PDAC from the TCGA, GTEx, and GEO database. Peritumoral tissue number vs tumor tissue number: TCGA and GTEx (171 vs 179), GSE71989 (8 vs 14), GSE43795 (5 vs 6), GSE101448 (19 vs 24), GSE62165 (13 vs 118), GSE28735 (45 vs 45, paired), GSE62452 (69 vs 69, paired), GSE32676 (7 vs 25). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, in different data sets. G Kaplan–Meier survival analysis of patients with different tumoral or peritumoral REG3A expression levels from the GSE28735 and GSE62452 data sets. H - P Single cells RNA sequencing analysis using CRA001160 data set. H UMAP representation of different subgroups; the expression levels of REG3A in each subgroup were plotted onto the UMAP. I Pseudo-time reconstitution of acinar and ductal cells with abnormal gene expression profiles and malignant ductal cells inferred by slingshot trajectory. J The KO/GO enrichment analysis between REG3A-positive and REG3A-negative acinar cells. K The heatmap view of trajectory in the slingshot trajectory. Color key indicates low to high expression levels. L The different cell clusters of the acinar and ductal cells. (M) The DEGs between REG3A-positive and REG3A-negative acinar cell clusters. N The expression of REG3A in different patients with PDAC from the CRA001160 cohort. O The up-regulated genes in the malignant ductal cells from the patients with higher acinar cell REG3A expression compared to patients with low acinar cell REG3A expression. P The GO/KEGG/Reactome pathway enrichment of up-regulated genes in ( O )

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: Expressing, Concentration Assay, Microarray, In Situ, RNA Sequencing, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: REG3A promotes in vitro and in vivo PDAC tumor growth. A The expression of REG3A in human pancreas tissue, rat AR42J cells, and human SW1990, PANC-1, AsPC-1, and BxPC-3 pancreatic cancer cell lines. B The PANC-1 cells were infected with a REG3A adenovirus overexpression vector (AdREG3A) or vector alone (AdVec), and expression of REG3A was determined by western blotting. All blots were repeated at least 3 times; tubulin was used as the internal reference. C At different times after infection, the cell viability of PANC-1 cells was measured. *** p < 0.001 compared to the Control group, ### p < 0.001 as indicated, n = 5. D After infection with 10^9 AdREG3A or AdVec for 1 week, PANC-1 cell colonies were stained with crystal violet, and the colony numbers were counted. E After infection with 10^9 AdREG3A or AdVec for 48 h, EdU staining was performed to assess the proliferative ratio in the different groups. F After infection with 10^9 AdREG3A or AdVec for 48 h, cell invasion was measured by transwell assay. G Photograph of PANC-1 xenograft tumors at 4 weeks after cell injection; The in vivo tumor volume was recorded after PANC-1 cell injection. *** p < 0.001 compared to the Control group, n = 5. H The xenograft tumor sections were stained with H&E dye, anti-REG3A, or anti-KI67 antibodies. Scale bar = 200 μm, and referred to all panels. I The concentration of REG3A in the culture medium 48 h after infection. *** p < 0.001 compared to the Control group, ### p < 0.001 as indicated, n = 5. J PANC-1 cells infected with 10^9 AdREG3A, AdVec, or REG3A sequence lacking the signal peptide (AdΔREG3A) and immunostained with anti-REG3A (red) or anti-Calnexin (green) antibodies; nuclei were stained with DAPI. Scale bar = 50 μm, and refers to all panels. K After infection with 10^9 AdREG3A, AdVec, or AdΔREG3A for 48 h, the concentration of REG3A in the culture medium was measured. *** p < 0.001 compared to the Control group, n = 6. L - O After infection, the cell viability, colony formation, EdU staining, invasion of PANC-1 cells were determined, ** p < 0.01, *** p < 0.001 compared to the AdVec group, n = 5

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: In Vitro, In Vivo, Expressing, Infection, Over Expression, Plasmid Preparation, Western Blot, Control, Staining, Transwell Assay, Injection, Concentration Assay, Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Recombinant REG3A stimulates PANC-1 cell growth via EGFR-MAPK pathways. A - D PANC-1 cells were treated with different concentrations of recombinant human REG3A (rhREG3A) for different durations and the cell viability, colony formation, EdU staining, and cell invasion were determined, ** p < 0.01, *** p < 0.001 in 1 μg/mL group, # p < 0.05, ## p < 0.01, ### p < 0.001 in 10 μg/mL group, compared to the vehicle-treated group, n = 5. E PANC-1 cells were treated with 10 μg/mL rhREG3A for 24 h, total RNA was isolated and RNA sequencing was performed. F Volcano plot showing the upregulated and downregulated genes in PANC-1 cells treated with 10 μg/mL rhREG3A for 24 h. G Heatmap showing the representative upregulated genes in rhREG3A-treated PANC-1 cells. H GO/KEGG enrichment analysis showing upregulated pathways in rhREG3A-treated PANC-1 cells. I Protein–protein interaction (PPI) analysis was performed using upregulated genes in the epidermal growth factor (EGF) pathway. J Gene set enrichment analysis (GSEA) of the upregulated genes in rhREG3A-treated PANC-1 cells. K Representative GSEA curves in ( J )

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: Recombinant, Staining, Isolation, RNA Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: EGFR-MAPK activation is involved in the proliferative effect of REG3A in PDAC cells. A Phosphokinase array analysis of PANC-1 cells serum starved for 24 h, followed by 10 μg/mL rhREG3A or vehicle treatment for 30 min. Dot blots are shown, with altered dots marked by red frames. B Phosphorylation of tyrosine kinase and C phosphorylation of EGFR and ERK detected by western blots of PANC-1 cells serum starved for 24 h, followed by 10 μg/mL rhREG3A treatment for different times. D Dimerization assay performed using the BS3 cross-linker, followed by western blot analysis using EGFR antibody in PANC-1 cells serum starved for 24 h, followed by 10 μg/mL rhREG3A or 100 ng/mL EGF treatment for 30 min. All blots were repeated at least 3 times; tubulin was used as an internal reference; the integrated optical density (IOD) of each band in the independent blot was analyzed (* p < 0.05, *** p < 0.001, compared to 0-time, n = 3). E The PANC-1 cells were treated with vehicle or 10 μg/mL rhREG3A for 1 h, then the cells were lysed for Co-immunoprecipitation (Co-IP). F Double immunofluorescent staining of REG3A (red) and EGFR (green), in vehicle or rhREG3A-simulated PANC-1 cells, DAPI (blue) was used to stain nuclei, white marker indicated the co-localization of REG3A and EGFR (yellow), scale bar = 20 μm. G The in vitro binding activity of rhREG3A to the extracellular domain of EGFR protein by MST assay

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining, Marker, In Vitro, Binding Assay, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: REG3A activates EGFR by direct binding to the extracellular domain of EGFR. A CHO cells were transfected with human EGFR or vector plasmids, and the expression of EGFR was confirmed. B CHO cells transfected with EGFR plasmid (CHO-EGFR) were starved for 24 h, and stimulated with 10 μg/mL rhREG3A for different times and evaluated for phosphorylation of EGFR and ERK by western blotting. C All blots were repeated at least 3 times; tubulin was used as an internal reference; the integrated optical density (IOD) of each band in the independent blot was analyzed (* p < 0.05, ** p < 0.01, *** p < 0.001, compared to 0-time, n = 3). D Schematic representation of the three-dimensional structure of a predicted REG3A–EGFR complex (illustrated with PyMOL). Red, full length of human REG3A; Cyan, the extracellular domain of EGFR (PDB ID: 1MOX). E Ligplot + diagram showing the protein residues that interact between REG3A (chain B) and EGFR (chain A). F Interaction plots for a predicted REG3A–EGFR complex. Residue colors: Gray, aliphatic (Ile, Ala, Leu, Val), blue, alkaline (Arg, Lys), orange (Gly, Pro), violet, aromatic (Tyr, Phe), yellow (Cys), red (Asp), green (Gln, Ser, Asn, Thr). Plot of H-bonds (blue lines), salt bridges (red lines), and non-bonded contacts (orange tick marks) between residues on either side of the REG3A-EGFR complex interface. G Schematic representation of the three-dimensional structure of a predicted EGFR ligand–EGFR complex (illustrated with PyMOL). Red, full length of different human EGFR ligands; Cyan, the extracellular domain of EGFR (PDB ID: 1MOX)

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Phospho-proteomics, Western Blot, Residue

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: REG3A binds to EGFR-ECD region, requiring its C-type lectin domain. A Schematic diagram of full-length (FL), C-terminal deletion (△C), N-terminal deletion (△N) constructs of Flag-REG3A; FL, extracellular domain (ECD), intracellular domain (ICD) of His-EGFR; signal peptide (SP), transmembrane (TM), juxtamembrane (JM) regions, kinase domain (KD), C-terminal region (CR). B , C After the CHO cells were co-transfected with different REG3A/EGFR truncations, anti-His antibody immunoprecipitation (IP: His) was performed. Representative immunoblot of EGFR-His and REG3A-Flag in IP and in whole-cell lysate (input) is shown. D The in vitro binding activity of rhREG3A to the ECD or the ICD of EGFR protein by MST assay, n = 3. E The in vitro binding activity of rhREG3A to the ECD of EGFR protein in the presence/absence of 10 ng/mL EGF by MST assay, n = 3. F The PANC-1 cells were starved for 24 h, and treated with 10 ng/mL EGF in the present/absent of 10 μg/mL rhREG3A for 5 min, or different times, the phosphorylation of EGFR and ERK were determined by western blotting. All blots were repeated at least 3 times; tubulin was used as an internal reference; the integrated optical density (IOD) of each band in the independent blot was analyzed (** p < 0.01, *** p < 0.001, as indicated, n = 3)

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: Construct, Transfection, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Activity Assay, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Inflammation-mediated STAT3 activation contributes to the up-regulated REG3A expression in acinar cells. A PySCENIC analysis of activated transcriptional factors, B The up-regulated mRNAs of transcriptional factors in REG3A-positive acinar cells compared to REG3A-negative acinar cells in CRA001160 dataset. C The intersection of ( A ), ( B ), and the transcriptional factors with binding sites with the promoter region of REG3A predicted by JASPAR database. D The REG3A expression in Fig. L. E The mRNA expression of STAT3 in Fig. L. F The representative image of REG3A and phosphorylated STAT3 expression in the TMA cohort. G The relationship between peritumoral REG3A and phosphorylated STAT3 expression level in the TMA cohort. H The transcriptional activation of STAT3 prediction by PySCENIC analysis in Fig. L. I The transcriptional activation of STAT3 between REG3A-positive acinar cells and REG3A-negative acinar cells in CRA001160 dataset. J The IL6-JAK-STAT3 pathway activation of STAT3 prediction by PySCENIC analysis in Fig. J. K The relationship between REG3A expression and STAT3 activation in ( J ). L The intersection of STAT3-positive and REG3A-positive acinar cells. M The IL6-JAK-STAT3 pathway activation between REG3A-positive acinar cells and REG3A-negative acinar cells in CRA001160 dataset. N The survival outcomes between different levels of IL6-JAK-STAT3 pathway activation. O The binding sites of STAT3 in the promoter region of REG3A predicted by JASPAR. P The luciferase reporter gene assay under IL6-stimulation, n = 3. Q The AR42J cells were stimulated with different concentration of IL6 in the presence/absence of 10 μM Stattic for 24 h, the protein was extracted and the expression levels of phosphorylated STAT3, total STAT3, REG3A, and Tubulin were measured by western blot analysis, all blots were performed at least for three times. R The AR42J cell lines were stimulated with different concentration of IL6 in the presence/absence of 10 μM Stattic for 24 h, the mRNA level of REG3A was detected by qPCR assay, *** p < 0.001, n = 4

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: Activation Assay, Expressing, Binding Assay, Luciferase, Reporter Gene Assay, Concentration Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: REG3A secreted by peritumoral acinar cells enhances pancreatic ductal adenocarcinoma progression via activation of EGFR signaling

doi: 10.1186/s12964-025-02103-4

Figure Lengend Snippet: Peritumoral acinar cell secreted REG3A activates EGFR-MAPK signal in PDAC. A The HE staining and immunohistochemical staining using REG3A, CK7, and KI67 antibodies in a same PDAC tissue with different magnifications. B Pathway enrichment in patients with high expression of REG3A in CRA001160 dataset. C Immunohistochemical staining of pERK and pEGFR in patients with different expression of REG3A (left) and vector or REG3A transfected PANC1 xenografts in nude mice (right). E Cell communications between different cell clusters in REG3A Low and REG3A High patients in CRA001160 dataset. F Graphic abstract. Inflammatory signals-stimulated STAT3 activation contributes to the increased REG3A expression in acinar cells, which then the secreted REG3A stimulates the growth of PDAC cell by activating EGFR signal directly

Article Snippet: Briefly, the proteins were firstly incubated with anti-REG3A antibodies for 2 h and then mixed with Protein G beads overnight (Beyotime).

Techniques: Staining, Immunohistochemical staining, Expressing, Plasmid Preparation, Transfection, Activation Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Quantification of REG3A mRNA expression in the skin wounds of normal or diabetic patients with acute injury ( n =5). ( b ) Immunohistochemical analysis of REG3A in the skin wounds from three normal or diabetic patients. The samples from normal patients were taken 1 day (two patients) or 2 days after acute injury, whereas the samples from diabetic patients were taken 5 or 9 or 14 days after acute injury. ( c ) The expression of RegIIIγ mRNA in skin wounds of normal and T1D mice 3 days after aseptic injury ( n =6). ( d ) Immunoblot analysis of RegIIIγ in skin extracts taken from 2 mm surrounding the wound edges of normal and T1D mice. ( e ) Immunohistochemical analysis of RegIIIγ in day-3 skin wounds of normal and T1D mice. ( f ) Quantification of TNF-α and IL-6 mRNA expression in day-3 skin wounds from normal and T1D mice treated with or without 100 μg RegIIIγ ( n =8). ( g ) Quantification of TNF-α and IL-6 production by ELISA in day-3 skin wounds of normal and T1D mice ( n =5). ( h ) Wound healing of normal and T1D mice treated with ( n =7) or without 100 μg RegIIIγ ( n =6). ( i ) Photographs of healing wounds from normal and T1D mice treated as in h . All the mice used in the study without specific notes were C57BL/6 strain, and T1D mice were developed by STZ induction. Long scale bars represent 200 μm. Short scale bars represent 50 μm. Black rectangles designate region of × 400 magnification shown in inset of b and e . The abbreviations used here are normal (Nor), diabetes mellitus (DM), unwounded (unW), wounded (W) and RegIIIγ (R). * P <0.05, ** P <0.01 and *** P <0.001. P values were determined by two-tail t -tests ( a ) or two-way analysis of variance (ANOVA; c , f – h ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Expressing, Immunohistochemical staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) IL-17 production by ELISA in skin extracts taken from 2 mm skin surrounding the wound edges of normal and T1D mice at indicated times ( n =4). ( b ) Immunoblot of REG3A in NHEKs stimulated by different doses of rhIL-33 for 12 h. ( c ) RegIIIγ and IL-33 production by ELISA in skin extracts taken as in a ( n =4). ( d , e ) Immunohistochemical analyses of IL-33 in day-3 skin wounds of normal and T1D mice ( d ) or skin wounds of normal and diabetic patients taken as in ( e ). Long scale bars represent 200 μm while short scale bars represent 50 μm. Black rectangles designate region of × 400 magnification shown in inset. ( f ) Immunoblot of RegIIIγ in the skin wounds of T1D mice treated with PBS or rmIL-33. The first two samples were day-3 skin wounds of normal and T1D mice. The other three samples were from the skin wounds of T1D mice treated with rmIL-33 a day before wounding (−1) or 1 or 3 days post wounding. ( g ) Wound healing of normal and T1D mice treated with ( n =7) or without rmIL-33 ( n =6). ( h ) IL-33 production in NHEKs induced by different doses of rhIL-17 for 24 h. ( i ) The production of IL-33 and RegIIIγ in day-3 skin wounds of wild-type (WT) and Il17 −/− mice. ( j ) REG3A production in NHEKs induced by 200 ng ml −1 rhIL-17 before and after IL-33 was silenced. ( k ) Immunoblot of RegIIIγ in skin wounds of WT and Il17 −/− mice treated with PBS or 2 μg rmIL-33. ( l ) IL-33 production induced by 200 ng ml −1 rhIL-17 in NHEKs exposed to 20 mM glucose or mannitol for 24 h. ( m ) Immunofluorescent staining of IL-33 in NHEKs treated as in l . Scale bars represent 25 μm. ( n ) IL-33 production in NHEKs treated with 200 ng ml −1 rhIL-17 in the presence or absence of 20 mM glucose before and after AGE was inhibited by 2 mM aminoguanidine. The abbreviations used here are IL-33 shRNA ( IL-33 sh), glucose (Glu), mannitol (Man), aminoguanidine (AG). * P <0.05, ** P <0.01 and *** P <0.001. P values were determined by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemical staining, Staining, shRNA

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Quantification of Tlr3 mRNA expression in day-3 skin wounds of normal and T1D mice ( n =6). ( b ) Quantification of TNF-α and IL-6 mRNA expression in the skin extracts taken from 2 mm surrounding the wound edges of WT and Tlr3 −/− T1D mice at day 3 ( n =6). ( c ) Wound healing of normal WT ( n =12), normal Tlr3 −/− ( n =8), diabetic WT ( n =12) and diabetic Tlr3 −/− ( n =10) mice. ( d ) Quantification of TNF-α ( n =5) and IL-6 ( n =8) protein by ELISA in the skin extracts taken from 2 mm surrounding the wound edges of WT and Tlr3 −/− T1D mice treated with or without RegIIIγ. ( e ) Quantification of TNF-α and IL-6 protein by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A ( n =3). ( f ) Quantification of TNF-α and IL-6 protein by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) in the presence or absence of different mutants of REG3A ( n =3). ( g ) Quantification of TNF-α and IL-6 protein by ELISA in adult human epidermal keratinocytes (AHEKs) treated with 10 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A or C-REG3A ( n =3). * P <0.05, ** P <0.01 and *** P <0.001. NS, no significance; ND, not detected. P values were analysed by one-way analysis of variance (ANOVA; f , g ) or two-way ANOVA ( a – e ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Immunoblot of SHP-1 in NHEKs stimulated with 30 nM REG3A for indicated times. ( b ) Immunoblot of SHP-1 in AHEKs stimulated with different doses of REG3A for 24 h. ( c ) Immunoblot of SHP-1 in mouse skin intradermally injected with 100 μg RegIIIγ for indicated times. ( d , e ) Immunoblot of SHP-1 in NHEKs transfected with different doses of pSG5-C-REG3A ( d ) for indicated times ( e ). ( f ) Immunoblot of SHP-1 in NHEKs stimulated with 100 ng ml −1 rhIL-33 before and after REG3A silencing. ( g ) TNF-α and IL-6 mRNA expression in NHEKs transfected with 1 μg pCMV vector or pCMV-SHP-1 in the presence or absence of 5 μg ml −1 poly(I:C) for 24 h ( n =3). ( h ) TNF-α and IL-6 mRNA expression in NHEKs treated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after SHP-1 silencing ( n =3). ( i ) TNF-α and IL-6 production in AHEKs treated as in h ( n =3). ( j ) TNF-α and IL-6 mRNA expression in day-3 skin wounds of WT normal or T1D mice ( n =5). The mice were intradermally injected with H 2 O or 4 mg of SHP-1 inhibitor SSG before wounding. Three days later, 2 mm skin surrounding the wounds was taken for RNA isolation. The abbreviations used here are SHP-1 shRNA ( SHP-1 sh) and REG3A (R). * P <0.05, ** P <0.01 and *** P <0.001. NS, no significance; ND, not detected. P values were analysed by one-way analysis of variance (ANOVA; g ) or two-way ANOVA ( h – j ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Western Blot, Injection, Transfection, Expressing, Plasmid Preparation, Isolation, shRNA

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Interaction between Flag-tagged different domains of EXTL3 and REG3A assessed by immunoblot analysis after immunoprecipitation with anti-Flag or anti-REG3A. M: mock; F: full-length EXTL3; N: N-EXTL3 (1–548); C: C-EXTL3 (141–919); ΔNΔC: EXTL3 ΔNΔC (141–548). ( b ) Interaction between REG3A or C-REG3A and Flag-tagged N-EXTL3 assessed by immunoblot analysis after immunoprecipitation with anti-REG3A or anti-Flag. F: full-length REG3A; C: CTLD domain of REG3A. ( c ) Quantification of TNF-α and IL-6 mRNA expression in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after EXTL3 silencing ( n =3). ( d ) Immunoblot of SHP-1 in NHEKs treated with 30 nM REG3A before or after EXTL3 silencing. EXTK3 si: EXTL3 siRNA. ( e ) SHP-1 production in NHEKs treated with 30 nM REG3A in the presence or absence of NF-κB inhibitor (Bay11, 10 μM), Erk inhibitor (PD98059, 20 μM), p38 MAPK inhibitor (SB202190, 5 μM), PI3K inhibitor (LY294002, 50 μM), STAT3 inhibitor (S3I-201,50 μM) and AKT inhibitor (AKT1/2 inhibitor, 8 μM). ( f ) Immunoblot of phosphorylated AKT in NHEKs treated with 30 nM REG3A for 1 h before or after EXTL3 was silenced. ( g ) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or AKT1/2 inhibitor for 1 h. AKT1/2 i: AKT1/2 inhibitor. ( h ) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or STAT3 inhibitor (S3I-201) for 1 h. STAT3 i: STAT3 inhibitor. *** P <0.001. NS, no significance. P values were analysed by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a , b ) Quantification of TNF-α and IL-6 production by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) ( a ) or in AHEKs treated with 10 μg ml −1 poly(I:C) ( b ) in the presence or absence of JNK inhibitor (SP600125, 15 μM) for 24 h ( n =3). ( c ) The phosphorylation of JNK and c-Jun in NHEKs stimulated by 5 μg ml −1 poly(I:C) in the presence or absence of JNK inhibitor for 1 h. JNK i: JNK inhibitor. ( d , e ) Normalized luciferase activity in 293T cells co-transfected with the plasmid expressing c-Jun (0, 50, 100, 150, 200 ng) and plasmids containing TNF-α promoter (50 ng; d ), or TNF-α promoter with mutation in c-Jun binding site (50 ng; d ), or IL-6 promoter (100 ng; e ), or IL-6 promoter with the mutation in c-Jun binding site (100 ng; e ) for 24 h. ( f ) The phosphorylation of JNK and c-Jun in WT primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( g ) The phosphorylation of JNK2 and c-Jun in Jnk1 −/− primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( h ) The phosphorylation of JNK1 and c-Jun in Jnk2 −/− primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( i , j ) JNK2 phosphorylation in the skin wounds of WT and Tlr3 −/− normal mice ( i ) or T1D mice ( j ). ( k ) Quantification of TNF-α and IL-6 production by ELISA in day-3 skin wounds of WT and Jnk2 −/− normal and T1D mice ( n =6). ( l ) Wound healing of WT ( n =6), Jnk1 −/− ( n =4) and Jnk2 −/− T1D mice ( n =6). ( m , n ) JNK2 phosphorylation in NHEKs stimulated by 5 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A ( m ) or C-REG3A ( n ). ( o ) JNK2 phosphorylation in AHEKs stimulated by 10 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A. * P <0.05, ** or ## P <0.01 and ***or ### P <0.001. P value was analysed by one-way analysis of variance (ANOVA; a , b ) or two-way ANOVA ( d , e , k , l ). Data are the means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Binding Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Phosphorylated JNK2 in NHEKs transfected with pCMV vector, pCMV-muSHP-1(C453S) and pCMV-SHP-1 in the presence or absence of 5 μg ml −1 poly(I:C). ( b ) Phosphorylated JNK2 in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after SHP-1 silencing. ( c ) Interaction between SHP-1 and JNK2 in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A assessed by immunoblot analysis after immunoprecipitation with anti-SHP-1 or anti-JNK2. ( d ) Immunoblot of p-JNK2, SHP-1 and RegIIIγ in the skin extracts taken from 2 mm surrounding the wound edges of WT normal and T1D mice at indicated times. ( e ) Immunoblot of p-JNK2, SHP-1 and RegIIIγ in the skin wounds of WT normal mice injected with IgG or RegIIIγ-neutralizing antibody. ( f ) Immunoblot of p-JNK2 and SHP-1 in the skin wounds of WT normal and T1D mice injected with 100 μg RegIIIγ. ( g ) Wound healing in T1D mice treated with or without 100 μg RegIIIγ before or after SHP-1 was inhibited by its inhibitor SSG ( n =9). ( h ) The schematic graph reflects the interaction between REG3A and TLR3 signalling. After skin injury, IL-33 induces REG3A expression in epidermal keratinocytes. REG3A, in turn, acts on keratinocytes to induce the negative regulator SHP-1 to selectively inhibit TLR3-activated JNK2, thus controlling TLR3-induced inflammation in skin wounds. However, in diabetes, hyperglycaemia inhibits IL-33 expression induced by IL-17. The reduction of IL-33 leads to the decrease in REG3A and SHP-1 but increased TLR3-activated JNK2 phosphorylation, thus exacerbating inflammation in diabetic skin wounds. The abbreviations used here are control (Ctrl), poly(I:C) (P), REG3A (R) and Reg Ab (RegIIIγ antibody). * P <0.05, and *** P <0.001. NS, no significance. P value was analysed by two-way analysis of variance (ANOVA). Data are the means±s.e.m. and representative of two independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Injection, Expressing, Phospho-proteomics, Control