Journal: Journal of cellular physiology

Article Title: RNA-binding protein PUM2 regulates mesenchymal stem cell fate via repression of JAK2 and RUNX2 mRNAs

doi: 10.1002/jcp.29281

Figure Lengend Snippet: A. Western blot was performed to analyse the protein levels of JAK2, RUNX2, PUM1, and PUM2 in mock- or PUM2-overexpressing, vector-transfected MSCs. The protein expression level of α-TUBULIN was used as a loading control. Each experiment was conducted using MSCs from at least three donors.

Article Snippet: The membranes were incubated for approximately 10 h with antibodies against PUM1 (GeneTex, Inc., CA, USA), PUM2 (human; Abcam, Cambridge, UK), PUM2 (zebrafish; Biorbyt Ltd, Cambridge, UK), JAK2 (EMD Millipore, San Diego, CA, USA), OCN (Abcam), RUNX2 (EMD Millipore), PPARγ (EMD Millipore), AP2 (Abcam), and FLAG (Sigma).

Techniques: Western Blot, Plasmid Preparation, Transfection, Expressing

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: a PUM1 mRNA expression levels in PAAD tissues (T) and normal tissues (N) analyzed by gene expression profiling interactive analysis (GEPIA). * P < 0.05. b Analysis of the relationship between PUM1 mRNA expression and overall survival of PAAD patients analyzed using The Cancer Genome Atlas (TCGA)

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Expressing, Gene Expression

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: a Representative images of PUM1 expression by immunohistochemistry (IHC) analysis in tissue microarrays representing 60 PDAC patients. b Comparison of total scores for PUM1 assessment on tissue microarrays slides. Total score = positive staining scores × staining intensity score. * P < 0.05. c The overall survival rate of patients with PDAC was evaluated based on PUM1 expression using Kaplan–Meier analysis ( n = 60, P = 0.022)

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Comparison, Staining

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: The relationship between PUM1 protein levels and clinic characteristics of patients with pancreatic adenocarcinoma

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: PUM1 mRNA ( a ) and protein ( b ) expression levels in PDAC cell lines (BxPC-3, AsPC-1, MIA PaCa-2, and PANC-1) and human pancreatic duct epithelial cells HPDE6-C7. c PUM1 expression levels in cells infected with lentivirus expressing the shRNA of PUM1 (shPUM1) or negative (NC) control empty lentivirus. d , e Effect of PUM1 knockdown on the OD values at 490 nm. f Effect of PUM1 knockdown on early apoptosis rate. b , c , f The representative graphs are on the left, and the statistical results are on the right. n = 3, * P < 0.05, for a – f

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Expressing, Infection, shRNA, Control, Knockdown

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: Effect of PUM1 knockdown on cell migration ( a ) and invasion ( b ) detected by Transwell assays. Representative graphs (left). Bar graph showing the statistical result of migratory or invasive cells (right). c Effect of PUM1 knockdown on the expression of MMP9, VEGF, vimentin, and E-cadherin. Representative graphs of western blotting (left). Relative protein expression in reference to GAPDH (right). n = 3, * P < 0.05, for a – c

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Knockdown, Migration, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: a PUM1 expression levels in tumors formed by the shPUM1 or NC group cells. b , c Effect of PUM1 knockdown on the growth of subcutaneous xenograft tumor. b Pictures of subcutaneous xenograft tumor. c Tumor growth curve. n = 8, * P < 0.05. d , e Effect of PUM1 knockdown on lung metastases examined by hematoxylin and eosin staining, or Ki67 expression detected by immunohistochemistry in the lung tissues of a lung metastasis mouse model

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Expressing, Knockdown, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: Volcano plot ( a ), scatter plot ( b ), and cluster gram ( c ) of the cDNA microarrays. d Effect of PUM1 knockdown on the expression of key components of the eIF2 signaling pathway. Left: Representative graphs of western blotting. Right: Statistical result of the relative protein expression in reference to GAPDH. n = 3, * P < 0.05

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Knockdown, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: PUM1 knockdown prevents tumor progression by activating the PERK/eIF2/ATF4 signaling pathway in pancreatic adenocarcinoma cells

doi: 10.1038/s41419-019-1839-z

Figure Lengend Snippet: a Representative pictures showing p-PERK levels in 60 PDAC tissues on tissue microarray examined by immunohistochemical analysis. b Linear regression analysis showing the correlation of PUM1 and p-PERK levels in PDAC tissues ( n = 60)

Article Snippet: The PUM1 primary antibody was purchased from ABclonal Technology (Wuhan, China).

Techniques: Microarray, Immunohistochemical staining

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: PUM1 was downregulated in MASLD. (A–F) PUM1 and PUM2 expression in the livers of MASLD patients and normal controls in the GSE126848 dataset (n=14 with normal control, n=16 with MASLD patients), GSE24807 dataset (n=5 with normal control, n=12 with MASLD patients), and GSE17470 dataset (n=4 with normal control, n=7 with MASLD patients). (G) Representing immunofluorescent images and fluorescence quantification of PUM1 and PUM2 expression in the liver tissues of MASLD patients and healthy controls (n=3). (H) Immunofluorescence staining showing Albumin (green) and PUM1 (red) colocalization in human liver tissues. (I, J) The representative images of HE staining of liver in mice. (K, L) Western blot analysis for PUM1 and PUM2 in the livers of mice (n=5). (M, O) Relative Pum1 expression in the livers of MASLD mice (n=5). (N, P) Correlation analysis of relative Pum1 expression with NAS score in MASLD mice. *, **, and *** denote p values <0.05, 0.01, and 0.001, respectively, in comparison to Normal groups or control groups. The white arrow indicates the colocalization of Albumin with PUM1. Abbreviations: CDAHFD, choline-deficient, l -amino acid–defined, high-fat diet; HE, hematoxylin and eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; NAS, NAFLD activity score; WD, western diet.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: Expressing, Control, Fluorescence, Immunofluorescence, Staining, Western Blot, Comparison, Activity Assay

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: PUM1 knockdown aggravated liver injury in WD+CCl 4 mice. (A) Schematic diagram for knockdown of PUM1 in WD+CCl 4 -induced MASLD model mice. (B) Body weight change curve of mice. (C–E) Serum ALT or AST concentrations and liver index in mice (n=10). (F) Western blot analysis for PUM1 expression in liver, heart, lung, kidney, brain, and testicle in MASLD mice. (G) Western blot analysis for PUM1 expression in mouse liver (n=4). (H) Immunofluorescence staining showing Albumin (green) and PUM1 (red) colocalization in mouse liver tissues. (I) The representative images of HE staining, Oil red O staining, Sirius red staining, and F4/80 staining of liver in mice. (J–N) HE + area, Oil red O + area, Sirius red + area, NAS score, and liver fibrosis scores of mice in 3 groups (n=10). The white arrow indicates the colocalization of Albumin with PUM1. The black arrow indicates ballooning hepatocytes. *, **, and *** denote p values <0.05, 0.01, and 0.001, respectively, in comparison to control groups. #, ##, and ### denote p values <0.05, 0.01, and 0.001, respectively, in comparison to the WD+CCl 4 +shNC group. Liver index=liver weight/body weight × 100%. HE + area (%)=area of fat vacuole/entire sample area × 100%. Oil red O + area (%)=area of Oil red O staining/entire sample area × 100%. Sirius red + area (%)=area of Sirius red staining/entire sample area × 100%. Abbreviations: HE, hematoxylin and eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; NAS, NAFLD activity score; WD, western diet.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence, Staining, Comparison, Control, Activity Assay

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: PUM1 knockdown aggravated liver injury in CDAHFD mice. (A) Schematic diagram for knockdown of PUM1 in CDAHFD-induced MASLD model mice. (B) Body weight change curve of mice. (C–E) Serum ALT or AST concentrations and liver index in mice (n=10). (F) Western blot analysis for PUM1 expression in liver, heart, lung, kidney, brain, and testicle in MASLD mice. (G) Western blot analysis for PUM1 expression in mouse liver (n=4). (H) Immunofluorescence staining showing Albumin (green) and PUM1 (red) colocalization in mouse liver tissues. (I) The representative images of HE staining, Oil red O staining, Sirius red staining, and F4/80 staining of liver in mice. (J–N) HE + area, Oil red O + area, Sirius red + area, NAS score, and liver fibrosis scores of mice in 3 groups (n=10). The white arrow indicates the colocalization of Albumin with PUM1. The black arrow indicates ballooning hepatocytes. ***denotes p values <0.001 in comparison to control groups. #, ##, and ### denote p values <0.05, 0.01, and 0.001, respectively, in comparison to CDAHFD+shNC groups. Liver index=liver weight/body weight × 100%. HE + area (%)=area of fat vacuole/entire sample area × 100%. Oil red O + area (%)=area of Oil red O staining/entire sample area × 100%. Sirius red + area (%)=area of Sirius red staining/entire sample area × 100%. Abbreviations: CDAHFD, choline-deficient, l -amino acid–defined, high-fat diet; HE, hematoxylin and eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; NAS, NAFLD activity score.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence, Staining, Comparison, Control, Activity Assay

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: PUM1 regulated lipid deposition and lipotoxic death in steatotic hepatocytes. (A) Western blot analysis for PUM1 in PA-treated HepG2 cells, primary mouse hepatocytes, macrophages, and LX-2 cells (n=3). (B, C, F, G) Western blot analysis for PUM1, SREBP1, ACC1, and FASN expression in the steatosis hepatocytes treated with si-PUM1 or oe-PUM1 (n=3). (D, H) Representative images of BODIPY staining of steatotic hepatocytes treated with si-PUM1 or oe-PUM1. (E, I) PI staining of steatotic hepatocytes treated with si-PUM1 or oe-PUM1 (n=3). (J) Western blot analysis for PUM1, SREBP1, ACC1, and FASN expression in the HepG2 cells and primary mouse hepatocytes (n=3). (K, L) Representative images of BODIPY staining of HepG2 cells and primary mouse hepatocytes. (M, N) PI staining of HepG2 cells and primary mouse hepatocytes (n=3). *, **, and *** denote p values <0.05, 0.01, and 0.001, respectively, in comparison to the BSA, si-NC, and oe-Vector groups. Abbreviations: BSA, bovine serum albumin; FASN, fatty acid synthase; PA, palmitic acid; PI, propidium iodide.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: Western Blot, Expressing, Staining, Comparison, Plasmid Preparation

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: Hepatocyte-derived exosomes regulated the activation of HSCs via PUM1. (A) Transmission electron microscopy, (B) nanoparticle tracking analysis, and (C) western blot analysis were used to characterize HepG2 cell–derived exosomes. (D, E) Western blot analysis for αSMA, COL1A1, and COL3A1 expression in normal LX-2 cells or PUM1-knockdown LX-2 cells (n=3). (F, I) Transmission electron microscopy, (G, J) nanoparticle tracking analysis, and (H, K) western blot analysis were used to characterize exosomes from primary mouse hepatocytes. (L, M) Western blot analysis for αSMA, COL1A1, and COL3A1 expression in primary mouse HSCs (n=3). (N) Transmission electron microscopy, (O) nanoparticle tracking analysis, and (P) western blot analysis were used to characterize primary mouse hepatocyte-derived exosomes. (Q, R) Western blot analysis for αSMA, COL1A1, and COL3A1 expression in normal JS-1 cells or PUM1-knockdown JS-1 cells (n=3). (S) Transmission electron microscopy, (T) nanoparticle tracking analysis, and (U) western blot analysis were used to characterize exosomes from PUM1-knockdown primary mouse hepatocytes. (V) Western blot analysis for αSMA, COL1A1, and COL3A1 expression in normal JS-1 cells (n=3). *, **, and *** denote p values <0.05, 0.01, and 0.001, respectively, in comparison to BSA-exo, Control-exo, or Normal-exo. Abbreviations: BSA, bovine serum albumin; CDAHFD, choline-deficient, L-amino acid–defined, high-fat diet; Exo, exosomes; PA, palmitic acid; WD, western diet.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: Derivative Assay, Activation Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Knockdown, Comparison, Control

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: PUM1 regulates HSC activation. (A) The expression of PUM1 in HSCs in the GSE128940 dataset (n=3). (B) The relative mRNA expression of PUM1 in LX-2 cells in our in vitro experiments (n=3). (C, D) Western blot analysis for PUM1 expression in LX-2 cells (n=3). (E) Heatmap of RNA sequencing analysis for the expression of fibrosis-related genes in LX-2 cells. (F, I) Western blot analysis for COL1A1, COL3A1, and αSMA expression in LX-2 cells (n=3). (G, J) Relative mRNA levels of PUM1 , αSMA , and COL3A1 in LX-2 cells (n=3). (H, K) Representing immunofluorescent images of αSMA expression in LX-2 cells. (L, M) Western blot analysis for COL3A1 and αSMA expression in the liver of MASLD mice (n=4). ** and *** denote p values <0.01 and 0.001, respectively, in comparison to control, si-NC groups or oe-Vector groups. #, ##, and ### denote p values <0.05, 0.01, and 0.001, respectively, in comparison to the WD+CCl 4 +shNC group or CDAHFD+shNC groups. Abbreviations: CDAHFD, choline-deficient, l -amino acid–defined, high-fat diet; MASLD, metabolic dysfunction–associated steatotic liver disease; WD, western diet.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: Activation Assay, Expressing, In Vitro, Western Blot, RNA Sequencing, Comparison, Control, Plasmid Preparation

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: PUM1 targeted TPM4 mRNA in HSCs. (A) The volcano map of RNA sequencing analysis for differentially expressed genes in LX-2 cells. (B) The heatmap of RNA sequencing analysis for differentially expressed genes in LX-2 cells. (C) The intersection of the results of RNA sequencing analysis and the predicted results in the catRAPID database. (D, E) Relative mRNA expression of TPM4 , CHST14 , and SMIM10L2B in LX-2 cells (n=3). (F, G) Western blot analysis for TPM4 expression in LX-2 cells (n=3). (H, I) Western blot analysis for TPM4 in the livers of MASLD mice (n=5). (J) The binding sites of PUM1 protein and TPM4 mRNA from the catRAPID database. (K) RNA immunoprecipitation (RIP) experiment for validation of the binding sites of PUM1 protein and TPM4 mRNA (n=3). (L) Western blot analysis for TPM4 expression in LX-2 cells (n=3). (M, N) Western blot analysis for PUM1 and TPM4 expression in LX-2 cells (n=3). *, **, and *** denote p values <0.05, 0.01, and 0.001, respectively, in comparison to control groups, si-NC groups, or oe-Vector groups. Abbreviations: CDAHFD, choline-deficient, l -amino acid–defined, high-fat diet; MASLD, metabolic dysfunction–associated steatotic liver disease; WD, western diet.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: RNA Sequencing, Expressing, Western Blot, Binding Assay, RNA Immunoprecipitation, Biomarker Discovery, Comparison, Control, Plasmid Preparation

Journal: Hepatology Communications

Article Title: Hepatocyte-derived Pumilio1-enriched exosomes inhibit HSC activation by suppressing tropomyosin-4 translation

doi: 10.1097/HC9.0000000000000759

Figure Lengend Snippet: PUM1 targeted TPM4 to regulate HSCs activation. (A, B) RT-qPCR analysis and western blot analysis for the overexpression effect of TPM4 in LX-2 cells (n=3). (C, D) RT-qPCR analysis and Western blot analysis for αSMA, COL1A1, and COL3A1 in LX-2 cells (n=3). (E) Representing immunofluorescence images for αSMA in LX-2 cells. (F, G) RT-qPCR analysis and Western blot analysis for TPM4 expression in LX-2 cells (n=3). (H, I) RT-qPCR analysis and Western blot analysis for αSMA, COL1A1, and COL3A1 in LX-2 cells (n=3). (J, L) Representing immunofluorescence images for αSMA in LX-2 cells. (K) Western blot analysis for COL1A1, αSMA, PUM1, and TPM4 in LX-2 cells (n=3). *, **, and *** denote p values <0.05, 0.01, and 0.001, respectively, in comparison to si-NC or oe-Vector groups. ### denote p values <0.001, respectively, in comparison to si-TPM4 groups. Abbreviation: RT-qPCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: Cell lysates were then immunoprecipitated overnight with anti-PUM1 antibody (1:20, #49885, Signalway Antibody) or IgG at 4 °C.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Over Expression, Immunofluorescence, Expressing, Comparison, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction