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: 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 Scheme of caerulein injection to induce acute pancreatitis. Acute pancreatitis was induced by intraperitoneal (i.p.) injection with caerulein dissolved in PBS at a dose of 50 μg/kg at hourly intervals eight times daily, for two consecutive days. PBS injection alone served as control. b The size of pancreas of PBS-treated mice appear smaller than those of caerulein-treated mice ( n = 6, student’s t -test). c Percentage of pancreas/body weight of caerulein-treated mice versus PBS-treated mice ( n = 6, student’s t -test). d Abundant ADM formation in caerulein-treated mice (H&E, 40×, Scale bar: 500 μm). e Difference in the extent of ADM between PBS-treated mice and caerulein-treated mice, as measured by percentage of ADM area in total pancreatic area on H&E stained tissue sections ( n = 6, student’s t -test). f Western blot analysis with quantifications showed higher REG3B protein level and lower AMYLASE protein level in the pancreatic tissue of caerulein-treated mice than that of PBS-treated mice ( n = 6, student’s t -test). GAPDH served as a loading control. g RT-qPCR analysis showed higher Ck19 and lower Mist1 mRNA levels in the pancreatic tissue of caerulein-treated mice than that of PBS-treated mice ( n = 5, student’s t -test). h H&E staining of caerulein-induced ADM in mice, with corresponding REG3B IHC staining. ADM areas were highlighted by dashed lines, with arrows indicating naïve ADM and asterisks indicating relatively mature ADM. Scale bar: 200 μm. Data are represented as means ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001; Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Injection, Control, Staining, Western Blot, Quantitative RT-PCR, Immunohistochemistry, 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 Scheme of intraperitoneal injection of different reagents in the five experimental groups. b At day 10 (D10), no ADM phenotype was observed in WT mice injected with caerulein. WT mice injected with both recombinant mouse REG3B protein and caerulein, and the REG3B TG mice injected with caerulein showed persistent ADM as indicated by the ductal morphology (black arrow heads) in H&E staining. Focal PanIN (marked by black asterisk) was observed in two out five REG3B TG mice injected with caerulein. Scale bar: 200 μm. c WT mice injected with recombinant REG3B and caerulein and the REG3B TG mice injected with caerulein demonstrate higher levels of Ck19 and lower levels of Mist1 mRNA. ( n = 5, one-way ANOVA). d Immunofluorescence staining shows gain of CK19 expression (red) and loss of AMYLASE (green) in the ADM area of WT mice injected with recombinant REG3B and caerulein and in the Reg3b TG mice injected with caerulein. Scale bars: 20 μm. White arrows indicate ADM. e Persistence of ADM in WT mice injected with recombinant REG3B 2, 4, and 10 weeks after caerulein injection. ( n = 5, magnification ×200, scale bar: 200 μm). Values in graphs are means ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001. Non-significant (n.s.) if P > 0.05. n = 5 per group, WT wild type, TG REG3B transgenic mice, Cae caerulein.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Injection, Recombinant, Staining, Immunofluorescence, Expressing, IF-P, Transgenic Assay

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

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: a Western blot analysis of related protein expression in 266-6 and AR42J cell lines 30 min and 72 h after different stimulations, with quantification data. b , c Expression level of JAK2/STAT3 signaling components in the 266-6 cell line under different conditions for 30 min ( b ) and 72 h ( c ). d , e Expression level of JAK2/STAT3 signaling components in the AR42J cell line under different conditions for 30 min ( d ) and 72 h ( e ) ( n = 3, one-way ANOVA test). REG3B treatment alone did not increase p-JAK nor p-STAT3 expression at two timepoints in two cell lines but did increase their expression once the receptor EXTL3 was blocked.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Western Blot, 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: 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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Membrane, Mutagenesis

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

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: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Membrane, Mutagenesis