anti pum1  (Bethyl)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Prolyl endopeptidase is a multifunctional host factor required for FMDV infection

doi: 10.1007/s00018-025-05941-0

Figure Lengend Snippet: PREP -KO IBRS-2 cells upregulate RIG-I mRNA expression by decreasing PUM1 stability. A Western blot analysis of the expression levels of key molecules involved in the type I IFN signaling pathway in PREP -KO and sgNC IBRS-2 cells. B Western blot analysis of RIG-I protein expression levels in GFP-PREP overexpression and WT IBRS-2 cells. C RT-qPCR analysis of relative RIG-I , MDA5 and MAVS mRNA expression levels in PREP -KO and sgNC IBRS-2 cells. D RT-qPCR analysis of relative PUM1 mRNA expression levels in PREP -KO and sgNC IBRS-2 cells. E Western blot analysis of PUM1 protein expression levels in PREP -KO and sgNC IBRS-2 cells. F RT-qPCR analysis of the relative mRNA expression levels of IFN-β , RIG-I , MDA5 and OASL in IBRS-2 cells transfected with either control siRNA (siNC) or PUM1-targeting siRNA (siPUM1). G Co-immunoprecipitation analysis of lysates from HEK293T cells co-transfected with PREP-Myc and PUM1-Flag. H Western blot analysis of PUM1 expression levels in PREP -KO and sgNC IBRS-2 cells treated with CHX (100 µg/mL) for the indicated times. I Western blot analysis of PUM1 expression levels in PREP -KO and sgNC IBRS-2 cells maintained in the presence of DMSO, MG132 (4 or 20 µM), CQ (20 or 50 µM) or Z-VAD-FMK (10 or 50 µM). J Western blot analysis of PUM1 expression levels in PREP -KO and sgNC IBRS-2 cells maintained in the presence of DMSO, Z-DEVD-FMK (15 µM), Z-IETD-FMK (15 µM) or Z-LEHD-FMK (15 µM). K Dual-luciferase reporter analysis of relative porcine RIG-I promoter activity in IBRS-2 cells transfected with siPUM1 or siNC, followed by transfection with porcine RIG-I dual-luciferase reporter plasmids. sgNC, sgRNA negative control; KO, knock out; WT, wild-type; kDa, kilodaltons. Data are represented as means ± SD. n = 3 ( C , D , F and K ). * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, no significant. P values were determined by two-tailed Student′s t -tests

Article Snippet: Membranes were blocked with 5% skim milk for 1 h and then probed with primary antibodies: PREP (Bethyl, #A305-348 A, 1:1,000), β-actin (Proteintech, #66009-lg, 1:2,000), PUM1 (Proteintech, #26256-1-AP, 1:1,000), VP1 (prepared in our laboratory, 1:1,000), 3D (prepared in our laboratory, 1:1,000), RIG-I (prepared in our laboratory, 1:1,000), TBK1 (Cell Signaling Technology, #3504, 1:1,000), IRF3 (Cell Signaling Technology, #4302, 1:1,000), IκBα (Cell Signaling Technology, #4812, 1:1,000), p65 (Cell Signaling Technology, #4764, 1:1,000), Myc (Cell Signaling Technology, #2278, 1:1,000), Flag (Cell Signaling Technology, #2368, 1:1,000).

Techniques: Expressing, Western Blot, Over Expression, Quantitative RT-PCR, Transfection, Control, Immunoprecipitation, Luciferase, Activity Assay, Negative Control, Knock-Out, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Prolyl endopeptidase is a multifunctional host factor required for FMDV infection

doi: 10.1007/s00018-025-05941-0

Figure Lengend Snippet: PREP is a multifunctional host factor required for FMDV infection. In IBRS-2 cells, PREP interacts with PUM1, while PREP -KO reduces the stability of PUM1. The destabilization of PUM1 enhances porcine RIG-I promoter activity, upregulating RIG-I and MDA5, thereby activating the type I IFN signaling pathway. In other FMDV-susceptible cells, PREP -KO inhibits the FMDV life cycle at the replication stage

Article Snippet: Membranes were blocked with 5% skim milk for 1 h and then probed with primary antibodies: PREP (Bethyl, #A305-348 A, 1:1,000), β-actin (Proteintech, #66009-lg, 1:2,000), PUM1 (Proteintech, #26256-1-AP, 1:1,000), VP1 (prepared in our laboratory, 1:1,000), 3D (prepared in our laboratory, 1:1,000), RIG-I (prepared in our laboratory, 1:1,000), TBK1 (Cell Signaling Technology, #3504, 1:1,000), IRF3 (Cell Signaling Technology, #4302, 1:1,000), IκBα (Cell Signaling Technology, #4812, 1:1,000), p65 (Cell Signaling Technology, #4764, 1:1,000), Myc (Cell Signaling Technology, #2278, 1:1,000), Flag (Cell Signaling Technology, #2368, 1:1,000).

Techniques: Infection, Activity Assay

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. PUM1&2 repression activity was measured in HCT116 cells using Nano-luciferase reporters with three PREs within a minimal 3′ UTR with cleavage and poly-adenylation signals (Nluc 3xPRE), calculated relative to a version wherein the PRE sequences were mutated (indicated in red text) to prevent PUM binding (Nluc 3xPREmt). PUM repression of the poly-adenylated reporter was compared to a Nluc reporter that has a 3′ end generated by the MALAT1 non-coding RNA (Nluc 3xPRE MALAT), which is processed by RNase P mediated cleavage to form a triple helix structure (PBD: 4PLX). Derivatives of the Nluc 3xPRE were constructed with internal poly(A) tracts of either 20 (A20) or 60 (A60) adenosines, inserted between the PREs and the MALAT1 triple helix. Firefly luciferase (Fluc) served as an internal control to normalize transfection efficiency. B. PRE dependent repression by endogenous PUM1 and PUM2 was measured as log fold change (FC) of each Nluc 3xPRE reporter relative to its corresponding 3xPREmt reporter. Mean fold change is plotted along with individual replicate data points. n=9; 3 experiments, each with 3 biological replicates; +/- standard deviation (SD). For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = **** based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Asterisks above the axis denote significance relative to the 3xPREmt version of each reporter type, whereas below the bars are calculated relative to the poly-adenylated Nluc reporters. C. Western blot confirming the depletion of PAN2 and CNOT1 proteins by RNAi in HCT116 cells. GAPDH served as a loading control. n=3 experimental replicates. D. The effect of CCR4-NOT or PAN2 and PAN3 knockdown on PUM repression of the Nluc 3xPRE reporter, relative to the mutant Nluc 3xPREmt, was measured in HCT116 cells, in comparison to cells transfected with non-targeting control siRNAs. n=9; 3 experiments, each with 3 biological replicates; +/- SD.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Activity Assay, Luciferase, Binding Assay, Generated, Construct, Control, Transfection, Standard Deviation, Western Blot, Knockdown, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. PABPC1 and PABPC4 co-immunoprecipitate with PUM1 from HCT116 cell extracts treated with RNases A and One. PUM1, eIF4E, eIF4G, PABPC1, PABPC4, and GAPDH were detected by western blot. B. Denaturing formaldehyde agarose gel analysis confirmed depletion of RNA in the HCT116 cell extracts before (-) or after treatment with RNase A and RNase One (+). The 18S and 28S ribosomal RNA bands, detected by ethidium bromide, are indicated on the right. C. Human PABPC1 domain architecture showing the N-terminal RRM domains (green) with the critical eIF4G binding site residues and the proline rich linker and C-terminal region PABC domain (blue) containing the MLLE motif residues important binding by PAM2-domain containing proteins. D. Western blot of the co-immunoprecipitation analysis of endogenous PUM1 with either HaloTag (HT), as a negative control, or HT-PABPC1 full-length (aa1-636) or HT-RRMs 1-4 (aa 1-370). All input samples were treated with RNase A and One. IgG beads and GAPDH served as negative controls. Dashed vertical lines in the panels indicate that the images were cropped to show relevant lanes. E. Western blot of PUM1 immunoprecipitates to detect association with either wild type full-length HT-PABPC1, or mutant versions wherein the eIF4G binding site is mutated (M161A and D165A) or the MLLE motif is mutated to (MLLEmt: M584G, L585A, L586A, and E587R), or the HT-MLLE domain (aa 542-636). All input samples were treated with RNase A and One.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Agarose Gel Electrophoresis, Binding Assay, Immunoprecipitation, Negative Control, Mutagenesis

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Western blot analysis confirms auxin induced depletion of degron-tagged PABPC1-AID and RNAi depletion of PABPC4 either individually or in combination in HCT116 cells. RNAi was performed by transfecting cells with either PABPC4 or non-targeting control NTC siRNAs. PABPC1-AID protein was depleted upon treatment with indole-3-acetic acid (+IAA), in comparison to vehicle only control (-IAA). GAPDH served as a loading control. B. PUM-mediated repression of Nluc 3xPRE reporter expression was measured in HCT116 cells, relative to the mutant PRE version. Individual and combined effects of PABPC4 RNAi and IAA induced depletion of PABPC1-AID on PUM repression were tested. n=9; 3 experiments, each with 3 biological replicates; +/- SD. For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = **** based on ordinary one-way ANOVA and Tukey test for multiple comparisons. C. Western blot analysis of samples from a representative experimental replicate for experiments shown in Panels D and E. GAPDH served as a loading control and PUM1 and PUM2 protein levels were detected as controls. The effect of combined PABPC4 RNAi and PABPC1-AID depletion on PUM repression of Nluc reporters containing the 3′ UTRs of the natural, PRE-containing, PUM target mRNAs FZD8 in panel D and CDKN1B in panel E were measured, relative to matched PRE mutant versions. n=9; 3 experiments, each with 3 biological replicates; +/- SD. Live cell numbers (panel F ) and viability (panel G ) were measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel B. n=12; 3 experiments, each with 2 biological replicates, 2 technical replicates; +/- SD.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Control, Comparison, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RTqPCR in RNA samples purified from HCT116 cells wherein PUM1 and PUM2 were depleted by RNAi. Changes in mRNA levels were determined relative to cells treated with the non-targeting control siRNAs (NTC). The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. B. Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RTqPCR in RNA samples purified from HCT116 cells wherein PABPC1-AID and PABPC4 were depleted by auxin treatment (+IAA) and RNAi, respectively. Changes in mRNA levels were determined relative to cells treated with vehicle only (-IAA) and the NTC siRNA. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. In both panels A and B, transcript level was measured by RTqPCR, normalized to 18S rRNA, and plotted as fold change relative to the non-depleted control condition. n=3 biological replicates, ± SD. For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****, ns = not significant based on unpaired two-tailed t-tests. C. Western blot analysis confirmed RNAi depletion of PUM1 and PUM2 in biological replicates in panel A. GAPDH served as a loading control. D. Western blot analysis of PABPC1-AID and PABPC4 confirmed their depletion by auxin induced degradation and RNAi, respectively, in samples from panel B. Histone H3 served as a loading control.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Expressing, Purification, Control, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Western blot analysis of halotag (HT) and HT-PABPC1 titration for samples from a representative experimental replicate of panel B. The amount of transfected plasmid for each effector is shown at the top. GAPDH served as a loading control. PUM1 and PUM2 were detected as an additional control. B. Reporter assay showing the effect of HT-PABPC1 over-expression on PUM repression of the Nluc 3xPRE reporter in wild type HCT116 cells, calculated relative to the Nluc 3xPREmt reporter. Halotag served as a negative control. n=9; 3 experiments, each with 3 biological replicates; +/- SD. For significance calling, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = **** based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Significance indicated above the X-axis indicates relative to 3xPREmt reporter, whereas significance calling shown below is indicated by the respective brackets. C. Graph of the fold change in HT-PABPC1 exogenous expression over endogenous PABPC1 levels calculated from three experimental replicates including the western blot shown in panel A. n=3; +/- SD. D. Western blot analysis of halotag (HT), HT-PABPC1 full-length, HT-PABPC1 full-length RNA- binding mutant (RBmt), HT-RRM1-4 domains, and HT-RRM1-4 RBmt samples taken from a representative experimental replicate of panel E. GAPDH served as a loading control. PUM1 and PUM2 were detected as controls. E. Reporter assay showing effect of HT-PABPC1 full-length, HT-RRM1-4 domains, and RNA- binding mutants versions when over-expressed on PUM repression of the Nluc 3xPRE reporter in HCT116 cells. n=9; 3 experiments, each with 3 biological replicates; +/- SD. Halotag served as a negative control. F. Western blot analysis of over-expressed HT-PABPC1 on PUM repression and the effect of the mutation of the eIF4G binding site mutant (M161A, D165A), or the MLLE motif (MLLEmt), or the RNA-binding defective mutant (RBmt) from a representative experimental replicate of panel G. H3 served as a loading control. PUM1 and PUM2 were detected as additional controls. G. Reporter assay showing effect of HT-PABPC1 full-length mutants on PUM repression in wild type HCT116 cells. n=9; 3 experiments, each with 3 biological replicates; +/- SD. Halotag served as a negative control.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Western Blot, Titration, Transfection, Plasmid Preparation, Control, Reporter Assay, Over Expression, Negative Control, Expressing, RNA Binding Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

doi: 10.1101/2025.10.02.680050

Figure Lengend Snippet: A. Co-immunoprecipitation analysis of PUM1 binding to the Nluc 3xPRE reporter in the presence of over-expressed halotag (HT) or HT-PABPC1. The top two panels show western blot detection of PUM1 protein and HT-PABPC1 in the 2% of the input HCT116 cell extracts the and 25% of the PUM1 RNA co-immunoprecipitates (RIP) from three biological replicate samples per condition. The bottom panel shows detection of Nluc mRNA by northern blotting. For northern blots, 1% of input samples and 75% of the RIP samples were loaded on the formaldehyde-MOPS agarose gel, respectively. Co-immunoprecipitation with IgG beads served as a negative control. B. Fold increase of the Nluc 3xPRE mRNA in the input samples from cells expressing HT- PABPC1 are plotted relative to the HT control, based on data in panel A. n=3 biological replicates, +/- SD. For significance calling, p < 0.001 = *** based on an unpaired student’s t-test. C. Fold enrichment of the Nluc 3xPRE mRNA in PUM1 RIP samples was measured relative to the IgG control. Importantly, the mRNA levels in each RIP sample was normalized to that present in the respective input samples. n=3 biological replicates, +/- SD. For significance calling, the difference between HT and HT-PABPC1 was not significant (p > 0.05 = ns) based on an unpaired student’s t-test.

Article Snippet: For each sample, 8.3 μl of protein A conjugated dynabeads (Thermo Fisher Scientific, 10001D) were washed 3 times with 250 μl 1xPBS + 0.1% Tween-20 and incubated in 100 μl of 1xPBS + 0.1% Tween-20 and 2 μg PABPC1 antibody (Abcam, ab6125) or PUM1 antibody (Bethyl labs, A302-576A) overnight, rotating at 4°C.

Techniques: Immunoprecipitation, Binding Assay, Western Blot, Northern Blot, Agarose Gel Electrophoresis, Negative Control, Expressing, 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 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