recombinant human reg3a his  (Sino Biological)

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: Cancer-free survival and overall survival of patients with cirrhosis or HCC, respectively, according to hepatic REG3A expression. (A) Kaplan-Meier curve of tumor-free survival (GSE15654; n = 216 patients with hepatitis C–related early-stage cirrhosis). Patients with high REG3A-expressing cirrhosis have longer tumor-free survival than those with low REG3A-expressing cirrhosis. (B) Overall survival of patients with HCC according to the level of REG3A expression in the tumor (TCGA; n = 286). (C, D) Overall survival of patients with HCC (GSE14520) according to the level of REG3A expression in (C) nontumor area adjacent to the tumor (HCC-NT; n = 209) and (D) in the tumor area (HCC-T; n = 221). No difference in overall survival was found in patients with HCC, whether or not the tumor area expressed REG3A. Abbreviations: NT, nontumor; REG3A, regenerating family member 3 alpha; T, tumor.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A strongly reduced cancer development in 2 mouse models of HCC. (A, B) WT and transgenic mice overexpressing REG3A in hepatocytes (REG3A-TG) received a single injection of the chemotoxic agent DEN at 14 days of age, and then tumor growth was monitored in independent groups of mice monthly for 8 months. (A) Proportion of mice with tumors over time. n = 9–16 mice for WT group and n = 9–14 for REG3A-TG group. (B) Left: Representative macroscopic views. Scale bar: 10 mm. Right: Circle pie charts for counting mice with healthy (dark green) or tumor livers (color-coded by nodule size, measured by the sum of the largest diameters of the nodule). WT livers show multiple nodules of (sub)centimeter size from 6 months of age. Transgenic livers are macroscopically normal over a longer time period or contain much smaller and fewer nodules than WT livers. The value shown in each portion of the pie chart represents the percentage of mice with a given nodule size. Arrows: the smallest tumor nodules detected at 6 months. (C–F) Genetic model for HCC in single transgenic mice for MYC (MYC-TG) and double transgenic mice for MYC and REG3A (MYC/REG3A-TG). (C) Liver weight to body weight (LW/BW). n = 12–20 independent mice per time and per group of mice. (D) Percentage of mice with HCC tumors over time (n = 40). (E) Left: Representative macroscopic views. Scale bar: 10 mm. Dotted lines: delineated tumor mass when possible. Right: Proportion of mice with healthy (dark green) or tumor (other colors of the code) liver. (F) Kaplan-Meier curve of overall survival of mice. Data are means ± SEM. The 1-tailed Fisher exact test was performed for analysis except for (C) (Student t test). * p < 0.05, ** p < 0.01. NS or no statistical significance, no significance. Abbreviations: DEN, diethylnitrosamine; REG3A, regenerating family member 3 alpha; WT, wild-type.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Transgenic Assay, Injection

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A is associated with a decrease in O -GlcNAcylation of MYC in mouse liver carcinoma. (A) qRT-PCR analysis of MYC transcript levels in liver samples at the indicated time points and pathological conditions. MYC-TG, transgenic mice homozygous for MYC; MYC/REG3A-TG, transgenic mice double homozygous for MYC, and REG3A. Healthy, normal liver sample from 3-month-old mice; preK, estimated precancerous liver sample from 6-month-old mice, livers in which the absence of tumor nodules was assessed macroscopically and microscopically; nontumor areas (NT); tumor areas (T); n = 6–16 per group. (B) Immunoblots for MYC and REG3A proteins and quantification of MYC from total protein extracts in REG3A-negative and positive tumors. Each dot represents a tumor sample from an independent mouse. (C) Immunoblots for the indicated proteins after nucleo-cytoplasmic fractionation and quantification of nuclear (Nuc) to cytosolic (Cyt) MYC ratio. (D) Quantification of total MYC protein levels and pT58 to total MYC ratio. (E) Immunoblots of phospho-GSK3β (Ser9), total GSK3β, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and total Erk1/2, and related densitometry. Each dot represents a sample from an independent mouse. (F) Succinylated WGA lectin pull down to determine the level of O -GlcNAcylated MYC in MYC tumors expressing or not REG3A. n = 3 independent experiments. The arrow indicates the REG3A signal. Asterix: nonspecific signal. (G) Immunoblots for O -GlcNAc and MYC proteins following immunoprecipitation of MYC from liver extracts of MYC and MYC/REG3A transgenic mice. The arrow indicates the MYC signal. n = 3 independent experiments. Data are averages ± SEM. The Mann-Whitney U test was performed for analysis except for (E) (paired Wilcoxon test with correction for multiple testing). NS or no statistical indication, no significance. Abbreviations: qRT-PCR, quantitative real-time PCR; REG3A, regenerating family member 3 alpha; WGA, wheat germ agglutinin.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Quantitative RT-PCR, Transgenic Assay, Western Blot, Fractionation, Expressing, Immunoprecipitation, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A alters the MYC-dependent transcriptional program in mouse HCC. (A–F) Differentially expressed genes identified by microarray analysis of mouse liver specimens expressing MYC (MYC-TG) or MYC and REG3A (MYC/REG3A-TG). n = 5 mice per group. Enrichment plots of genes related to the S3 subclass (A) or S1 subclass (B) of the molecular classification of Hoshida et al in murine MYC tumors expressing REG3A or not, respectively. (C, D) Pathway enrichment analysis showing the different molecular pathways from enrichment analysis of differentially expressed genes in nontumor area (C) and tumor area (D) of MYC-REG3A mouse livers compared with MYC mouse livers. (E) Enrichment plots of the 100 most expressed genes in human HCCs that express endogenous REG3A (TCGA data set). The top 100 signatures is enriched in HCCs from MYC/REG3A-TG mice compared to MYC-TG mice. (F) Enrichment plot of an MYC core signature (n = 77) described by Ji et al that is strongly attenuated in MYC tumors expressing REG3A compared to MYC tumors not expressing REG3A. Data are averages ± SEM. The Mann-Whitney U test was performed for analysis. NS or no statistical indication, no significance. Abbreviation: REG3A, regenerating family member 3 alpha.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Microarray, Expressing, MANN-WHITNEY

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: Preneoplastic and tumor livers of HCC expressing REG3A show substantial reduction of O -GlcNAcylation in mice and humans. Anti-RL2 immunoblots for O -GlcNAc. Proteins in nuclear (Nuc) and cytosolic (Cyt) fractions of (A) preneoplastic (preK) livers (n = 6), (B) in the nontumor area (n = 6) and (C) the tumor area (n = 12) of HCC from transgenic mice homozygous for MYC (MYC-TG) and transgenic mice double homozygous for MYC and REG3A (MYC/REG3A-TG). Quantification by densitometry below western blots. Each dot represents a sample from an independent mouse. (D) Western blots for O -GlcNAc proteins in livers of WT (n = 6) and transgenic mice overexpressing REG3A in the liver (REG3A-TG; n = 4) with DEN-induced HCC. (E) Western blots for O -GlcNAc proteins and endogenous REG3A protein in human HCC. Quantification of O -GlcNAc proteins in 18 NT and T matched samples. P1 = patient 1. Increased O -GlcNAcylation in 13 T samples (red line) and no detectable real change in O -GlcNAcylation in 5 T samples (green line) compared with matched NT samples ( p = 0.0047). (F) O -GlcNAcylation in human HCC samples as a function of whether they express or not endogenous REG3A in the NT and T areas. Ponceau S and Coomassie blue staining were used as loading controls. Data are averages ± SEM. The Mann-Whitney U test was performed except for (E) (paired Student t test) and (F) (Student t test). NS or no statistical indication, no significance. Abbreviations: DEN, diethylnitrosamine; NT, nontumor; REG3A, regenerating family member 3 alpha; T, tumor; WT, wild-type.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Expressing, Western Blot, Transgenic Assay, Staining, MANN-WHITNEY

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A bound to glucose and glucose intermediates is a regulator of protein glycosylation. (A) Quantification of HS and CS disaccharides by ion-pair reversed-phase chromatography in HuH7 cells expressing REG3A or EXTL3 alone, or both, or appropriate empty vectors. (B) Representative sugar slot blot of mono- and polysaccharides deposited at the indicated doses and hybridized with recombinant REG3A lectin. Lactose, mannan: positive control for REG3A binding. (C) Slot blot of indicated sugars in the presence of a full-length human recombinant REG3A protein (rcREG3A) preincubated with BSA or BSA + lactose (Glc-Gal). (D) Slot blot of indicated sugars in the presence of rcREG3A or a mutant recombinant REG3A protein (rcREG3A EPN/GPG ) proteins. (E) Immunoblots for O -GlcNAc (anti-RL2) and REG3A in nuclear fractions of HuH7 cells expressing REG3A, REG3A EPN/GPG or empty vector. Densitometry quantification (n = 3). (F) Enzymatic quantification of cellular UDP-GlcNAc content in preneoplastic (preK), nontumor (NT adjacent to a tumor), and tumor (T) liver samples from MYC/REG3A and MYC-TG transgenic mice. Each dot represents a sample of an individual mouse. (G) Rates of cellular ATP production in HuH7 cells expressing REG3A, REG3A EPN/GPG , or the empty vector, showing significant changes in total, mitochondrial, and glycolytic ATP production under the action of the full-length REG3A protein (n = 3). (H) Lectin blot for biotin-labeled PHA-L (phaseolus vulgaris leucoagglutinin) lectin on preneoplastic liver extracts from mice transgenic for MYC (MYC-TG) and for MYC and REG3A (MYC/REG3A-TG). Densitometry quantification (n = 6). (I) Glycogen concentrations in preneoplastic liver extracts (preK; left) from the mice shown (n = 6) and (J) in HuH7 cells expressing full-length REG3A or the mutant REG3A EPN/GPG (right) (n = 4). (K) Lectin blot for biotin-labeled WGA in extracts of preneoplastic (PreK) liver from MYC and MYC/REG3A transgenic mice and quantification by densitometry (n = 6). (L) A schematic view of carbohydrate metabolism from glucose and its intermediates. Gray = de novo synthesis of UDP-GlcNAc from glucose through the hexosamine synthesis pathway; Blue: salvage of GlcNAc through the metabolic processes that produce UDP-GlcNAc. *sugars bound to REG3A. Red arrows: carbohydrate pathways downregulated by REG3A. Equal symbols: glucose pathways not modulated by REG3A. Data are averages ± SEM. The Mann-Whitney U test was used for analysis, except for (E), (F), (G), and (J) (ANOVA test followed by a post hoc test). NS or no statistical indication, no significance. Abbreviations: CS, chondroitin sulfate; EXTL3, exostosin-like glycosyltransferase 3; HS, heparan sulfate; PHA-L, phytohemaglutinin-L; REG3A, regenerating family member 3 alpha; WGA, wheat germ agglutinin.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Glycoproteomics, Reversed-phase Chromatography, Expressing, Dot Blot, Recombinant, Positive Control, Binding Assay, Mutagenesis, Western Blot, Plasmid Preparation, Transgenic Assay, Labeling, MANN-WHITNEY

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: Cancer-free survival and overall survival of patients with cirrhosis or HCC, respectively, according to hepatic REG3A expression. (A) Kaplan-Meier curve of tumor-free survival (GSE15654; n = 216 patients with hepatitis C–related early-stage cirrhosis). Patients with high REG3A-expressing cirrhosis have longer tumor-free survival than those with low REG3A-expressing cirrhosis. (B) Overall survival of patients with HCC according to the level of REG3A expression in the tumor (TCGA; n = 286). (C, D) Overall survival of patients with HCC (GSE14520) according to the level of REG3A expression in (C) nontumor area adjacent to the tumor (HCC-NT; n = 209) and (D) in the tumor area (HCC-T; n = 221). No difference in overall survival was found in patients with HCC, whether or not the tumor area expressed REG3A. Abbreviations: NT, nontumor; REG3A, regenerating family member 3 alpha; T, tumor.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A strongly reduced cancer development in 2 mouse models of HCC. (A, B) WT and transgenic mice overexpressing REG3A in hepatocytes (REG3A-TG) received a single injection of the chemotoxic agent DEN at 14 days of age, and then tumor growth was monitored in independent groups of mice monthly for 8 months. (A) Proportion of mice with tumors over time. n = 9–16 mice for WT group and n = 9–14 for REG3A-TG group. (B) Left: Representative macroscopic views. Scale bar: 10 mm. Right: Circle pie charts for counting mice with healthy (dark green) or tumor livers (color-coded by nodule size, measured by the sum of the largest diameters of the nodule). WT livers show multiple nodules of (sub)centimeter size from 6 months of age. Transgenic livers are macroscopically normal over a longer time period or contain much smaller and fewer nodules than WT livers. The value shown in each portion of the pie chart represents the percentage of mice with a given nodule size. Arrows: the smallest tumor nodules detected at 6 months. (C–F) Genetic model for HCC in single transgenic mice for MYC (MYC-TG) and double transgenic mice for MYC and REG3A (MYC/REG3A-TG). (C) Liver weight to body weight (LW/BW). n = 12–20 independent mice per time and per group of mice. (D) Percentage of mice with HCC tumors over time (n = 40). (E) Left: Representative macroscopic views. Scale bar: 10 mm. Dotted lines: delineated tumor mass when possible. Right: Proportion of mice with healthy (dark green) or tumor (other colors of the code) liver. (F) Kaplan-Meier curve of overall survival of mice. Data are means ± SEM. The 1-tailed Fisher exact test was performed for analysis except for (C) (Student t test). * p < 0.05, ** p < 0.01. NS or no statistical significance, no significance. Abbreviations: DEN, diethylnitrosamine; REG3A, regenerating family member 3 alpha; WT, wild-type.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Transgenic Assay, Injection

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A is associated with a decrease in O -GlcNAcylation of MYC in mouse liver carcinoma. (A) qRT-PCR analysis of MYC transcript levels in liver samples at the indicated time points and pathological conditions. MYC-TG, transgenic mice homozygous for MYC; MYC/REG3A-TG, transgenic mice double homozygous for MYC, and REG3A. Healthy, normal liver sample from 3-month-old mice; preK, estimated precancerous liver sample from 6-month-old mice, livers in which the absence of tumor nodules was assessed macroscopically and microscopically; nontumor areas (NT); tumor areas (T); n = 6–16 per group. (B) Immunoblots for MYC and REG3A proteins and quantification of MYC from total protein extracts in REG3A-negative and positive tumors. Each dot represents a tumor sample from an independent mouse. (C) Immunoblots for the indicated proteins after nucleo-cytoplasmic fractionation and quantification of nuclear (Nuc) to cytosolic (Cyt) MYC ratio. (D) Quantification of total MYC protein levels and pT58 to total MYC ratio. (E) Immunoblots of phospho-GSK3β (Ser9), total GSK3β, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and total Erk1/2, and related densitometry. Each dot represents a sample from an independent mouse. (F) Succinylated WGA lectin pull down to determine the level of O -GlcNAcylated MYC in MYC tumors expressing or not REG3A. n = 3 independent experiments. The arrow indicates the REG3A signal. Asterix: nonspecific signal. (G) Immunoblots for O -GlcNAc and MYC proteins following immunoprecipitation of MYC from liver extracts of MYC and MYC/REG3A transgenic mice. The arrow indicates the MYC signal. n = 3 independent experiments. Data are averages ± SEM. The Mann-Whitney U test was performed for analysis except for (E) (paired Wilcoxon test with correction for multiple testing). NS or no statistical indication, no significance. Abbreviations: qRT-PCR, quantitative real-time PCR; REG3A, regenerating family member 3 alpha; WGA, wheat germ agglutinin.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Quantitative RT-PCR, Transgenic Assay, Western Blot, Fractionation, Expressing, Immunoprecipitation, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A alters the MYC-dependent transcriptional program in mouse HCC. (A–F) Differentially expressed genes identified by microarray analysis of mouse liver specimens expressing MYC (MYC-TG) or MYC and REG3A (MYC/REG3A-TG). n = 5 mice per group. Enrichment plots of genes related to the S3 subclass (A) or S1 subclass (B) of the molecular classification of Hoshida et al in murine MYC tumors expressing REG3A or not, respectively. (C, D) Pathway enrichment analysis showing the different molecular pathways from enrichment analysis of differentially expressed genes in nontumor area (C) and tumor area (D) of MYC-REG3A mouse livers compared with MYC mouse livers. (E) Enrichment plots of the 100 most expressed genes in human HCCs that express endogenous REG3A (TCGA data set). The top 100 signatures is enriched in HCCs from MYC/REG3A-TG mice compared to MYC-TG mice. (F) Enrichment plot of an MYC core signature (n = 77) described by Ji et al that is strongly attenuated in MYC tumors expressing REG3A compared to MYC tumors not expressing REG3A. Data are averages ± SEM. The Mann-Whitney U test was performed for analysis. NS or no statistical indication, no significance. Abbreviation: REG3A, regenerating family member 3 alpha.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Microarray, Expressing, MANN-WHITNEY

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: Preneoplastic and tumor livers of HCC expressing REG3A show substantial reduction of O -GlcNAcylation in mice and humans. Anti-RL2 immunoblots for O -GlcNAc. Proteins in nuclear (Nuc) and cytosolic (Cyt) fractions of (A) preneoplastic (preK) livers (n = 6), (B) in the nontumor area (n = 6) and (C) the tumor area (n = 12) of HCC from transgenic mice homozygous for MYC (MYC-TG) and transgenic mice double homozygous for MYC and REG3A (MYC/REG3A-TG). Quantification by densitometry below western blots. Each dot represents a sample from an independent mouse. (D) Western blots for O -GlcNAc proteins in livers of WT (n = 6) and transgenic mice overexpressing REG3A in the liver (REG3A-TG; n = 4) with DEN-induced HCC. (E) Western blots for O -GlcNAc proteins and endogenous REG3A protein in human HCC. Quantification of O -GlcNAc proteins in 18 NT and T matched samples. P1 = patient 1. Increased O -GlcNAcylation in 13 T samples (red line) and no detectable real change in O -GlcNAcylation in 5 T samples (green line) compared with matched NT samples ( p = 0.0047). (F) O -GlcNAcylation in human HCC samples as a function of whether they express or not endogenous REG3A in the NT and T areas. Ponceau S and Coomassie blue staining were used as loading controls. Data are averages ± SEM. The Mann-Whitney U test was performed except for (E) (paired Student t test) and (F) (Student t test). NS or no statistical indication, no significance. Abbreviations: DEN, diethylnitrosamine; NT, nontumor; REG3A, regenerating family member 3 alpha; T, tumor; WT, wild-type.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Expressing, Western Blot, Transgenic Assay, Staining, MANN-WHITNEY

Journal: Hepatology (Baltimore, Md.)

Article Title: Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway

doi: 10.1097/HEP.0000000000000993

Figure Lengend Snippet: REG3A bound to glucose and glucose intermediates is a regulator of protein glycosylation. (A) Quantification of HS and CS disaccharides by ion-pair reversed-phase chromatography in HuH7 cells expressing REG3A or EXTL3 alone, or both, or appropriate empty vectors. (B) Representative sugar slot blot of mono- and polysaccharides deposited at the indicated doses and hybridized with recombinant REG3A lectin. Lactose, mannan: positive control for REG3A binding. (C) Slot blot of indicated sugars in the presence of a full-length human recombinant REG3A protein (rcREG3A) preincubated with BSA or BSA + lactose (Glc-Gal). (D) Slot blot of indicated sugars in the presence of rcREG3A or a mutant recombinant REG3A protein (rcREG3A EPN/GPG ) proteins. (E) Immunoblots for O -GlcNAc (anti-RL2) and REG3A in nuclear fractions of HuH7 cells expressing REG3A, REG3A EPN/GPG or empty vector. Densitometry quantification (n = 3). (F) Enzymatic quantification of cellular UDP-GlcNAc content in preneoplastic (preK), nontumor (NT adjacent to a tumor), and tumor (T) liver samples from MYC/REG3A and MYC-TG transgenic mice. Each dot represents a sample of an individual mouse. (G) Rates of cellular ATP production in HuH7 cells expressing REG3A, REG3A EPN/GPG , or the empty vector, showing significant changes in total, mitochondrial, and glycolytic ATP production under the action of the full-length REG3A protein (n = 3). (H) Lectin blot for biotin-labeled PHA-L (phaseolus vulgaris leucoagglutinin) lectin on preneoplastic liver extracts from mice transgenic for MYC (MYC-TG) and for MYC and REG3A (MYC/REG3A-TG). Densitometry quantification (n = 6). (I) Glycogen concentrations in preneoplastic liver extracts (preK; left) from the mice shown (n = 6) and (J) in HuH7 cells expressing full-length REG3A or the mutant REG3A EPN/GPG (right) (n = 4). (K) Lectin blot for biotin-labeled WGA in extracts of preneoplastic (PreK) liver from MYC and MYC/REG3A transgenic mice and quantification by densitometry (n = 6). (L) A schematic view of carbohydrate metabolism from glucose and its intermediates. Gray = de novo synthesis of UDP-GlcNAc from glucose through the hexosamine synthesis pathway; Blue: salvage of GlcNAc through the metabolic processes that produce UDP-GlcNAc. *sugars bound to REG3A. Red arrows: carbohydrate pathways downregulated by REG3A. Equal symbols: glucose pathways not modulated by REG3A. Data are averages ± SEM. The Mann-Whitney U test was used for analysis, except for (E), (F), (G), and (J) (ANOVA test followed by a post hoc test). NS or no statistical indication, no significance. Abbreviations: CS, chondroitin sulfate; EXTL3, exostosin-like glycosyltransferase 3; HS, heparan sulfate; PHA-L, phytohemaglutinin-L; REG3A, regenerating family member 3 alpha; WGA, wheat germ agglutinin.

Article Snippet: They warmly thank Patricia Duchambon and her colleagues at the Unité d’Expression et de Purification des Protéines for the production of a recombinant human REG3A protein (CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris-Saclay, Orsay, France).

Techniques: Reversed-phase Chromatography, Expressing, Dot Blot, Recombinant, Positive Control, Binding Assay, Mutagenesis, Western Blot, Plasmid Preparation, Transgenic Assay, Labeling, MANN-WHITNEY

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