reg3a antibody  (Novus Biologicals)

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: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: REG3A expression is elevated in breast cancer tissues and cells. The Cancer Genome Atlas (TCGA) breast cancer cohort shows REG3A expression (RNA-Seq) in cancer tissues (“Tumor”, n = 1085) and normal breast tissues (“Normal”, n = 291) ( A ). Data from the GEO database (GSE9893 and GSE1379) demonstrates the correlation between REG3A expression and patient outcomes, including overall survival ( B ) and recurrence-free survival (RFS) ( C ).Expression of listed mRNAs and proteins in twenty ( n = 20) primary breast cancer tissues (“T”) and cancer-surrounding normal tissues (“N”) of local patients was shown ( D - F ). The representative REG3A immunohistochemistry (IHC) images of the described human tissues were shown ( G ). Expression of listed mRNAs and proteins in the described breast cancer cells and epithelial cells was shown ( H and I ). Values were mean ± standard deviation (SD). n = 5 for H and I. * P < 0.05 versus “Normal” tissues ( A ); * P < 0.05 versus “N” tissues ( D - F ); * P < 0.05 versus “pMEC” ( H and I ). Scale bar = 100 μm

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: Expressing, RNA Sequencing Assay, Immunohistochemistry, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: shRNA-induced knockdown of REG3A potently inhibits breast cancer cell proliferation and migration. The mRNA and protein expression of REG3A and REG1 in the stable pBC-1 primary breast cancer cells with the applied REG3A shRNA (“shREG3A-Sq2/3/6”, with non-overlapping sequences), the scramble control shRNA (“shC”), or in the parental control cells (“Ctrl”) was shown ( A and B ). Cells were further cultivated for indicated hours, cell proliferation (by measuring nuclear EdU incorporation, C ), cell migration ( D ) and invasion ( E ) were tested. The pBC-2 primary cancer cells, MCF-7 and MDA-231 lines, the primary mammary epithelial cells (pMEC) or established MCF-10A epithelial cells with shC and shREG3A-Sq6 were formed, and REG3A mRNA expression tested ( F and I ). Cells were further cultivated for designated hours, cell proliferation ( G and J ) and in vitro cell migration ( H ) were examined similarly. Values were mean ± standard deviation (SD, n = 5). * P < 0.05 versus “shC” group. “N. S.” stands for non-statistical difference ( P > 0.05). Experiments were repeated five times and similar results were obtained each time. Scale bar = 100 μm

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: shRNA, Migration, Expressing, In Vitro, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: shRNA-induced silencing of REG3A provokes apoptosis in breast cancer cells. The pBC-1 primary breast cancer cells with the applied REG3A shRNA (“shREG3A-Sq2/3/6”, with non-overlapping sequences), the scramble control shRNA (“shC”), as well as the parental control cells (“Ctrl”) were cultivated for indicated time periods, cell viability and death were tested by CCK-8 ( A ) and Trypan blue staining ( B ) assays, respectively; Caspase-PARP activation was examined as well ( C and D ); Levels of histone-bound DNA were measured via an ELISA assay ( E ), with mitochondrial depolarization tested by JC-1 fluorescence staining assay ( F ); Cell apoptosis was examined by nuclear TUNEL staining and TUNEL-positive nuclei percentage (% versus DAPI) was calculated ( G ). The pBC-1 primary breast cancer cells with shREG3A-Sq6 or shC were treated with z-DEVD-fmk (“zDEVD”, 30 µM), z-VAD-fmk (“zVAD”, 30 µM) or vehicle control (0.15% DMSO), cells were further cultivated for additional 96 h, cell viability (CCK-8 OD, H ) and death (Trypan blue staining assays, I ) were tested. The pBC-2 primary cancer cells, MCF-7 and MDA-231 established cancer cell lines, the primary mammary epithelial cells (pMEC) or established MCF-10A epithelial cells, with shC and shREG3A-Sq6, were cultivated for designated hours, cell viability (CCK-8 OD, J ), the caspase-3 activity ( K and M ) and cell apoptosis (by measuring TUNEL-positive nuclei percentage, L and N ) were tested. Values were mean ± standard deviation (SD, n = 5). * P < 0.05 versus “shC” group. # P < 0.05 ( H and I ). “N. S.” stands for non-statistical difference ( P > 0.05). Experiments were repeated five times and similar results were obtained each time. Scale bar = 100 μm

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: shRNA, CCK-8 Assay, Staining, Activation Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, TUNEL Assay, Activity Assay, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: REG3A knockout produces significant anti-breast cancer cell activity. The single stable pBC-1 cells, with the lentiviral CRISPR/Cas9-REG3A-KO construct (“koREG3A”) or the CRISPR/Cas9-control construct (“koC”), were established and expression of listed mRNAs and proteins was shown ( A and B ). Cells were further cultivated for indicated hours, cell proliferation (by measuring EdU incorporation, C ), cell migration ( D ) and invasion ( E ) were tested; Mitochondrial depolarization (JC-1 green monomers formation, F ); Caspase-3 activity ( G ) and cell apoptosis (TUNEL-positive nuclei percentage, H ) were measured as well. Values were mean ± standard deviation (SD, n = 5). * P < 0.05 versus “koC” group. “N. S.” stands for non-statistical difference ( P > 0.05). Experiments were repeated five times and similar results were obtained each time. Scale bar = 100 μm

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: Knock-Out, Activity Assay, CRISPR, Construct, Expressing, Migration, TUNEL Assay, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: REG3A overexpression results in cancer-promoting activity in breast cancer cells. The pBC-1 primary breast cancer cells expressing the REG3A-overexpressing construct (oeREG3A-Slc1 and oeREG3A-Slc2, representing two stable selections) or the empty vector (“Vec”) were established, and expression of listed mRNAs and proteins was shown ( A and B ). Cells were further cultivated for indicated hours, cell proliferation (by measuring EdU incorporation, C ), cell migration ( D ) and invasion ( E ) were tested; The pBC-2 primary breast cancer cells or established lines (MCF-7 and MDA-231) with the lentiviral REG3A-overexpressing construct (“oeREG3A”) or the empty vector (“Vec”) were established, and expression of REG3A mRNA was shown ( F ); Cells were further cultivated for designated time periods, cell proliferation ( G ) and migration ( H ) were tested similarly. Values were mean ± standard deviation (SD, n = 5). * P < 0.05 versus “Vec” group. “N. S.” stands for non-statistical difference ( P > 0.05). Experiments were repeated five times and similar results were obtained each time. Scale bar = 100 μm

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: Over Expression, Activity Assay, Expressing, Construct, Plasmid Preparation, Migration, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: REG3A is vital for Akt-mTOR activation in breast cancer cells. The pBC-1 primary breast cancer cells, with the applied REG3A shRNA (“shREG3A-Sq2/3/6”, with non-overlapping sequences), the scramble control shRNA (“shC”), the lentiviral CRISPR/Cas9-REG3A-KO construct (“koREG3A”), the CRISPR/Cas9-control construct (“koC”), REG3A-overexpressing construct (oeREG3A-Slc1 and oeREG3A-Slc2, two stable selections) or the empty vector (“Vec”), were cultured and expression of listed proteins was shown ( A - C ). The pBC-1 primary breast cancer cells with shREG3A-Sq6 were further stably transduced with the viral constitutively-active mutant S473D Akt1 (caAkt1) construct or the empty vector (“Vec”), expression of listed proteins was shown ( D ). Cells were further cultivated for designated hours, cell proliferation, migration and apoptosis were examined respectively through nuclear EdU staining ( E ), “Transwell” ( F ) and nuclear TUNEL staining ( G ) assays. The oeREG3A-Slc1 pBC-1 primary cells were treated with LY294002 or vehicle control (“Veh”, 0.15% DMSO) for designated hours, expression of listed proteins was shown ( H ); Cell proliferation ( I ) and migration ( J ) were tested using the same methods. Values were mean ± standard deviation (SD, n = 5). * P < 0.05 versus “shC”/“koC”/“Vec” group. # P < 0.05. Experiments were repeated five times and similar results were obtained each time. Scale bar = 100 μm

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: Activation Assay, shRNA, CRISPR, Construct, Plasmid Preparation, Cell Culture, Expressing, Stable Transfection, Transduction, Mutagenesis, Migration, Staining, TUNEL Assay, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: REG3A is important for maintaining the integrity of mTOR complexes. The pBC-1 primary breast cancer cells, with shREG3A-Sq6 (“shREG3A”), the lentiviral CRISPR/Cas9-REG3A-KO construct (“koREG3A”), REG3A-overexpressing construct (oeREG3A-Slc1 and oeREG3A-Slc2, two stable selections) or the empty vector (“Vec”), were cultured, mTOR-Raptor-Rictor associations were examined via co-immunoprecipitation (“IP”) assays ( A and B ), with expression of the listed proteins tested as “Inputs” ( A and B ). “Ctrl” stands for the parental control cells ( A ). Values were mean ± standard deviation (SD, n = 5). * P < 0.05 versus “Ctrl”/“Vec” group. Experiments were repeated five times and similar results were obtained each time

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: CRISPR, Construct, Plasmid Preparation, Cell Culture, Immunoprecipitation, Expressing, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: ZNF680 is a potential transcription factor of REG3A in breast cancer cells. The JASPAR database predicted potential transcription factors for REG3A ( A ). Following transfection of pBC-1 cells with the specified siRNAs targeting various transcription factors or a scramble non-sense siRNA (siC) for 48 h, the expression of REG3A mRNA was assessed ( B ). pBC-1 cells were then subjected to lentiviral ZNF680 shRNA (shZNF680-Sq1 or shZNF680-Sq2) and scramble control shRNA (“shC”) treatments ( C and D ), lentiviral CRISPR/Cas9-ZNF680-KO construct (“koZNF680”) and CRISPR/Cas9 control construct (“Cas9-C”) treatments ( E and F ), as well as lentiviral ZNF680-expressing construct (oeZNF680) or an empty vector (“Vec”) treatments ( G and H ), resulting in the establishment of stable cells. The expression of the listed mRNAs and proteins was then evaluated ( C - H ). Chromatin Immunoprecipitation (ChIP) assay results demonstrated the relative levels of ZNF680-bound REG3A promoter in specified breast tumor tissues (“T”) and matched adjacent normal tissues (“N”) ( I ) as well as in listed breast cancer cells and pMEC/MCF-10 A cells ( J ). “Ctrl” stands for the parental control cells. Values were mean ± standard deviation (SD). * P < 0.05 versus “siC” ( B ). * P < 0.05 versus “shC”/“Cas9-C”/“Vec” cells ( C - H ). * P < 0.05 versus “N” tissues or pMEC cells ( I and J ). Experiments were repeated five times and similar results were obtained each time

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: Transfection, Expressing, shRNA, CRISPR, Construct, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells

doi: 10.1186/s13058-024-01845-2

Figure Lengend Snippet: REG3A silencing impedes breast cancer xenograft growth in nude mice. The pBC-1 primary breast cancer cells, with REG3A shRNA (“shREG3A-Sq6”) or scramble control shRNA (“shC”), were subcutaneously ( s.c. ) injected to the flanks of the nude mice to form xenografts. The pBC-1 xenograft volumes ( A ) and animal body weights ( C ) were recorded starting at Day-22 (22 days after cell injection) and every six days after. At Day-56, all pBC-1 xenografts were isolated and weighted ( B ); At Day-32 and Day-44, one pBC-1 xenograft in each group was isolated and total four xenografts were obtained. Expression of listed mRNAs and proteins in fresh xenograft tissue lysates was tested ( D , E , F and J ); The relative caspase-3 activity was measured as well ( I ). Alternatively, pBC-1 xenograft slides were subject to immunohistochemistry (IHC) staining of pAkt (Ser-473) ( G ). Moreover, nuclear Ki-67/DAPI fluorescence staining ( H ) and nuclear TUNEL/DAPI fluorescence staining ( K ) were carried out in the described pBC-1 xenograft slides. Values were mean ± standard deviation (SD). In A - C , ten mice per group ( n = 10). For D - K , five random tissue pieces in each xenograft were tested. * P < 0.05 versus “shC” group. “N. S.” indicated no statistical difference ( P > 0.05). Scale bar = 100 μm

Article Snippet: The anti-REG3A antibody (ab202057) and anti-REG1 antibody (ab47099) were obtained from Abcam (Shanghai, China).

Techniques: shRNA, Injection, Isolation, Expressing, Activity Assay, Immunohistochemistry, Fluorescence, Staining, TUNEL Assay, Standard Deviation

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a H&E staining shows histological evidence of transformation from normal acini to ADM and to PDAC. Magnification, ×10; Scale bar: 2 mm. b Enlarged view of ADM area in a . Magnification, ×100; Scale bar: 200 μm. Black arrows indicate typical ADM circular structures. c Single antibody immunohistochemical staining shows intense REG3A expression the ADM zone. Magnification, ×10; Scale bar: 2 mm. d Enlarged view of ADM area in c . Black arrows indicate typical ADM stained strongly with REG3A. Magnification, ×100; Scale bar: 200 μm.

Article Snippet: Immunohistochemistry (IHC) was performed using primary antibodies against REG3A (1:150, R&D Systems, MAB5965), Amylase (1:100, Cell signaling technology) and CK19 (1:100, DSHB).

Techniques: Staining, Transformation Assay, Immunohistochemical staining, Expressing

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Bright field images showing an increase in ADM events (depicted by black arrows) in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (Magnification, ×200). TGFα-treated mouse primary acinar cells served as a positive control. b Bar graph showing increase in ADM quantity in 3D culture of mouse and human primary acinar cells during the 5-day REG3B or REG3A or TGFα treatment ( n = 3, 15 fields each group, one-way ANOVA and student’s t -test). c Bright field images in the upper row showing ADM events in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (magnification, ×630). Lower four rows are corresponding confocal immunofluorescence images showing a decrease in AMYLASE protein expression (green) and an increase in CK19 (red) protein in REG3B-induced, REG3A-induced, or TGFα-induced ADM (magnification, ×630. Scale bars: 20 μm). d RT-qPCR analysis showed a decrease in acinar-specific mRNA (Ptf1a, Cpa, and Mist1) and an increase in duct-specific mRNA (Ck19 and Nestin) in mouse and human primary acinar cells after 48 h of REG3B or REG3A treatment. ( n = 3 per group, student’s t -test). Values are represented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Immunohistochemistry (IHC) was performed using primary antibodies against REG3A (1:150, R&D Systems, MAB5965), Amylase (1:100, Cell signaling technology) and CK19 (1:100, DSHB).

Techniques: Cell Culture, Positive Control, Immunofluorescence, Expressing, Quantitative RT-PCR, Standard Deviation, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Western blot showing increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in the pancreatic tissue of REG3B-treated WT mice with caerulein-induced pancreatitis (WT cae+REG3B) and a moderate increase in TG mice with caerulein-induced pancreatitis (TG cae) ( n = 3, one-way ANOVA). b Western blots demonstrating increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in cultured human primary acinar cells and mouse acinar cell line 266-6 after REG3A or REG3B treatment respectively, for 48 h ( n = 3, student’s t -test). c Western blot showing the reduced expression of phosphorylated ERK, MEK, BRAF, and total KRAS in the 266-6 cell line after Reg3b gene knockdown by siRNA ( n = 3, student t -test). d In the upper panel, LY3009120 inhibited ERK, MEK, BRAF phosphorylation in a dose-dependent manner in 266-6 cell line. In the lower panel, LY3009120 (5 μM) blocked REG3B-induced MEK and ERK phosphorylation ( n = 3, one-way ANOVA). e Upper panel, Trametinib efficiently attenuated ERK phosphorylation in a dose-dependent manner in the AR42J cell line ( n = 3, one-way ANOVA). Lower panel, Trametinib (100 nM) blocked REG3B-induced ERK phosphorylation. f , g Bright field images ( g ) with quantification ( f ) show that Trametinib (100 nM) and LY3009120 (5 μM) treatment hindered REG3B-induced ADM in 3D cultures of mouse primary acinar cells (200×, n = 3, one-way ANOVA). h Confocal microscopy shows that Trametinib (100 nM) and LY3009120 (5 μM) treatment reduced CK19 protein expression (red) and increased AMYLASE protein expression (green) in 3D culture of mouse primary acinar cells (Scale bar: 20 μm). Data are represented as means ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Immunohistochemistry (IHC) was performed using primary antibodies against REG3A (1:150, R&D Systems, MAB5965), Amylase (1:100, Cell signaling technology) and CK19 (1:100, DSHB).

Techniques: Western Blot, Expressing, Cell Culture, Knockdown, Phospho-proteomics, Confocal Microscopy, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Co-localization of EXTL3 (green) with human REG3A (red) or rodent REG3B (red) in ADM zones derived from human primary acinar cells in 3D culture, and AR42J and 266-6 cell lines in 2D culture by immunofluorescence microscopy. (magnification: ×630, Scale bars: 10 μm). b Co-immunoprecipitation of REG3B and EXTL3 in mouse primary acinar cells, rat AR42J and mouse 266-6 acinar cell lines. Lysate-bead/antibody conjugate mixture was eluted with sample buffer without DTT for 10 min at 50 °C (elution 1). Sample buffer with DTT (100 mM) was added to the pelleted beads from elution 1 and boiled for 5 min (elution 2). c Confocal immunofluorescence microscopy showed that EXTL3 monoclonal antibody treatment effectively blocks REG3B-induced ADM as indicated by increased expression of the acinar marker AMYLASE and decreased expression of the ductal marker CK19. (magnification: ×630, Scale bar: 20 μm). d – f Extl3 siRNA or neutralizing antibody treatment reduced the protein expression of KRAS and phosphorylated ERK, MEK, and BRAF in the presence or absence of REG3B for 48 h. d Western blotting analysis of the knockdown of Extl3 by siRNA (20 nM) in 266-6 cell line, e knockdown of Extl3 by siRNA (20 nM) in the context of the 266-6 cell line treated with REG3B for 48 h, f Western blotting analysis of EXTL3 neutralizing antibody treatment (2 μg/ml) in 266-6 and AR42J cell lines and mouse primary acinar cells treated with or without REG3B for 48 h. Human primary acinar cells were treated for 30 min. TGFα treatment serves as a positive control for ADM induction.

Article Snippet: Immunohistochemistry (IHC) was performed using primary antibodies against REG3A (1:150, R&D Systems, MAB5965), Amylase (1:100, Cell signaling technology) and CK19 (1:100, DSHB).

Techniques: Derivative Assay, Immunofluorescence, Microscopy, Immunoprecipitation, Expressing, Marker, Western Blot, Knockdown, Positive Control

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: REG3B/REG3A binds to its receptor, EXTL3 receptor on the acinar cell membrane, and promotes ADM by activating the downstream RAS-RAF-MEK-ERK signaling pathway, in the absence of oncogenic Kras mutation. Targeting REG3B/REG3A, neutralizing its receptor EXTL3, or inhibiting downstream signaling molecules, such as B-RAF (LY3009120) or MEK1/2 (Trametinib), could interrupt the ADM process and potentially prevent early PDAC carcinogenesis.

Article Snippet: Immunohistochemistry (IHC) was performed using primary antibodies against REG3A (1:150, R&D Systems, MAB5965), Amylase (1:100, Cell signaling technology) and CK19 (1:100, DSHB).

Techniques: Membrane, Mutagenesis

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: FIGURE 1 | 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 30min 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: FIGURE 2 | 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 30min 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: FIGURE 3 | 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 × 105) 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 30min 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: FIGURE 4 | 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 30min 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: FIGURE 5 | 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 30min 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: FIGURE 6 | 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 × 105) 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 30min in a humidified chamber.

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