plc prf 5 plc  (ATCC)

Journal: Hepatology Communications

Article Title: Molnupiravir is effective against hepatitis E virus infection in an animal model

doi: 10.1097/HC9.0000000000000944

Figure Lengend Snippet: In vitro evaluation of molnupiravir in HEV cell culture models. (A) Cytotoxicity of molnupiravir, ribavirin, and sofosbuvir in PLC/PRF/5 cells and PRH. Three replicates per concentration. Bars represent the mean and SEM. Mean viability at each concentration was compared with the no-drug control group using the Student t test; ** indicates statistically significant difference ( p <0.05). (B) PRH infected with rHEV and treated with either PBS, varying concentrations of molnupiravir, ribavirin, and sofosbuvir, as well as combinations. (C) Immunofluorescence staining for rHEV-infected PRH with/without antivirals: uninfected cells (negative control), rHEV-infected cells treated with either PBS (no drug control), sofosbuvir 10 μM, ribavirin 200 μM, or molnupiravir 100 μM. Cells were stained with polyclonal anti-rHEV sera and counterstained with DAPI. (D) PLC/PRF/5 cells infected with HEV infectious clone Kernow-p6, (E) PLC/PRF/5 cells infected with human-derived HEV. Cells were treated with either PBS, varying concentrations of molnupiravir, ribavirin, and sofosbuvir, as well as combinations. Error bars represent SEM (n=3 replicates per concentration). Mean viral loads at each concentration were compared with the no-drug control group using the Student t test; * indicates statistically significant difference ( p <0.05) from the untreated mean. ** indicates an extremely statistically significant difference ( p <0.01) from the untreated mean. Abbreviations: HEV, hepatitis E virus; PRH, primary rat hepatocytes; SEM, standard error of the mean; PBS, phosphate-buffered saline; rHEV, Rocahepevirus ratti genotype 1; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Human hepatoma cell line PLC/PRF/5 (ATCC CRL-8024, LGC Standards, Wesel, Germany) and primary rat hepatocytes (PRH) were used in this study.

Techniques: In Vitro, Cell Culture, Concentration Assay, Control, Infection, Immunofluorescence, Staining, Negative Control, Derivative Assay, Virus, Saline

Journal: JHEP Reports

Article Title: H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression

doi: 10.1016/j.jhepr.2026.101786

Figure Lengend Snippet: Effects of environmental changes on histone lactylation in HCC. (A) Western blot analysis of H4K5la and H3K18la levels in HCC cell lines treated with high lactate concentrations (20 mM) and grown under glucose deprivation for 24 h panH4 was used as an internal control. (B) Effects of glycolytic inhibitor treatment with oxamate and 2-DG on H4K5la and H3K18la expression in HCC cells. (C) Experimental workflow employed to identify and validate genes associated with H4K5la and HCC malignancy. (D) Venn diagram based on RNA-seq data representing 13 commonly upregulated genes across all three cell lines with lactate treatment, as shown by overlaps. (E-F) Significantly upregulated pathways detected by GSEA for three HCC cell lines with lactate treatment. Normalized enrichment scores for key hallmark pathways, including hypoxia and glycolysis, in HLF, PLC/PRF/5, and SNU475 cells (E) and GSEA plots demonstrating the significant activation of the glycolysis pathway (F). 2-DG, 2-deoxyglucose; GSEA, gene set-enrichment analysis; H3K18la, histone H3 lysine 18 lactylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; panH4, total histone H4; RNA-seq, RNA sequencing.

Article Snippet: Three human HCC cell lines, HLF (RRID: CVCL_2947), PLC/PRF/5 (RRID: CVCL_0485), and SNU475 (RRID: CVCL_0497), were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, Expressing, RNA Sequencing, Activation Assay

Journal: JHEP Reports

Article Title: H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression

doi: 10.1016/j.jhepr.2026.101786

Figure Lengend Snippet: Functional analysis of EP300 as an H4K5la writer enzyme. (A) TCGA dataset analysis of mRNA expression levels of four HAT genes ( KAT5 , KAT6B , CREBBP , and EP300 ) in HCC. Red and black dots represent tumor and normal tissue samples, respectively. (B) Western blot analysis showing the effect of siRNA-mediated EP300 suppression (siEP300) on H4K5la levels in PLC/PRF/5 and HLF cells. Suppression of three additional HAT genes ( KAT5 , KAT6B , and CBP ) was also performed in HLF cells. (C) Effects of shRNA-mediated EP300 -KD in HCC cells. Stable cell lines were established using shEP300 #1, shEP300 #2, or negative control shRNA (NC) in two HCC cell lines. qRT-PCR analysis was performed to evaluate the KD efficiency of EP300. (D) Western blot analysis confirming the EP300 -KD and its effect on H4K5la levels in HCC cells. (E) qRT-PCR analysis showing the relative expression of four candidate genes in HCC cells with EP300 -KD in (shEP300 #1 and shEP300 #2) compared with shNC. (F) ChIP-qPCR analysis showing relative fold enrichment of H4K5la at the promoter regions of these four genes. Pan H4 and β-actin were used as internal controls for H4K5la and EP300 expression in Western blot analysis. 18S ribosomal RNA was used for qRT-PCR normalization. Relative levels of EP300 recruitment and H4K5la enrichment were normalized to Input DNA and pan H3, respectively, using ChIP-qPCR. Data are presented as the mean ± SD from three independent experiments, and statistical significance was determined using Student’s t test. Asterisks indicate statistical significance (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). ChIP, chromatin immunoprecipitation; H4K5la, histone H4 lysine 5 lactylation; HAT, histone acetyltransferase; HCC, hepatocellular carcinoma; KAT5, lysine acetyltransferase 5; KAT6B, lysine acetyltransferase 6B; NC, negative control; pan H4, total histone H4; qRT-PCR, quantitative reverse transcription PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA; TCGA, The Cancer Genome Atlas.

Article Snippet: Three human HCC cell lines, HLF (RRID: CVCL_2947), PLC/PRF/5 (RRID: CVCL_0485), and SNU475 (RRID: CVCL_0497), were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA).

Techniques: Functional Assay, Expressing, Western Blot, shRNA, Stable Transfection, Negative Control, Quantitative RT-PCR, ChIP-qPCR, Chromatin Immunoprecipitation, Reverse Transcription, Small Interfering RNA

Journal: JHEP Reports

Article Title: H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression

doi: 10.1016/j.jhepr.2026.101786

Figure Lengend Snippet: Positive feedback loop of glycolysis and ENO2 promotes H4K5la. (A, B) Western blot analysis showing the effects of ENO2 -KD and ENO2 -OE in HCC cells. Stable HCC cells with ENO2 -KD and ENO2 -OE were established using shRNAs (shENO2 #1 or shENO2 #2) (A) and an overexpression vector (B), respectively. Negative control shRNA (shNC) and GFP-expressing (Control) HCC cells served as controls in each experiment. pan H4 and β-actin were used as internal controls for H4K5la and ENO2 expression. (C, D) Intracellular concentrations of the key glycolytic metabolites and LDH activity in cell lysates of ENO2 -KD and ENO2 -OE HCC cells, and LDH activities. PEP was measured using a fluorescence assay with a microplate reader (excitation: 550 nm, emission: 590 nm). Lactate, pyruvate, and LDH activity were measured in cell lysates by a colorimetric assay with absorbance at 450 nm. (E, F) Relative expression levels of H4K5la-regulated genes and H4K5la/H4K5ac enrichment levels of their promoter regions in HCC cells with ENO2 -KD (E) and ENO2 -OE (F). (G) Re-expression of ENO2 in PLC/PRF/5 cells with ENO2 -KD using shENO2 #1 targeting the ENO2 3’-UTR. ENO2 -OE rescued global H4K5la expression as well as the expression and H4K5la enrichments at the promoters of three target genes. (H) ENO2/glycolysis/H4K5la positive feedback loop. β-actin and pan H4 were used as internal controls for Western blot analysis. 18S ribosomal RNA was used for qRT-PCR normalization. Relative H4K5la and H4K5ac enrichment levels were normalized to pan H4 by ChIP-qPCR. Data are presented as the mean ± SD from three independent experiments, and statistical significance was determined using Student’s t test. Asterisks indicate statistically significant differences (∗∗ p < 0.01, ∗∗∗ p < 0.001). ChIP, chromatin immunoprecipitation; H4K5ac, histone H4 lysine 5 acetylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; KD, knockdown; OE, overexpression; PEP, phosphoenolpyruvate; qRT-PCR, quantitative reverse transcription PCR; shNC, negative control short hairpin RNA; shRNA, short hairpin RNA.

Article Snippet: Three human HCC cell lines, HLF (RRID: CVCL_2947), PLC/PRF/5 (RRID: CVCL_0485), and SNU475 (RRID: CVCL_0497), were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Over Expression, Plasmid Preparation, Negative Control, shRNA, Expressing, Control, Activity Assay, Fluorescence, Colorimetric Assay, Quantitative RT-PCR, ChIP-qPCR, Chromatin Immunoprecipitation, Knockdown, Reverse Transcription

Journal: JHEP Reports

Article Title: H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression

doi: 10.1016/j.jhepr.2026.101786

Figure Lengend Snippet: Biological effects of ENO2 in vivo and clinical significance of ENO2 in HCC. (A–D) In vivo tumorigenicity of ENO2 -KD and its rescue models. A total of 1 × 10 6 PLC/PRF/5 cells with ENO2 -KD (shENO2 #1), ENO2 -KD with ENO2 re-expression (shENO2 #1 + ENO2 -OE), or negative control (shNC) were subcutaneously injected into two sites per mouse (bilateral flanks) in KSN/Slc nude mice (n = 6). Tumor growth was monitored for >26 days (A). Representative images of xenograft tumors (B) and final tumor volumes (C) are shown. D) IHC of ENO2, H4K5la, H4K5ac, and Ki67 in xenograft tumors. Re-expression of ENO2 restored the effects of ENO2 -KD. (E–H) In vivo therapeutic potential of the ENO2 inhibitor POMHEX. PLC/PRF/5 cells with ENO2 -OE were subcutaneously injected into KSN/Slc mice (n = 6, two injection sites per mouse), followed by treatment with POMHEX (40 mg/kg, i.p.) or vehicle (control) every 2 days (E: arrows indicate treatment). Final body weight (F), tumor volume (H), and photographs of xenograft tumors (G) are shown. (I) IHC of ENO2, H4K5la, H4K5ac, and Ki-67 in xenograft tumors treated with POMHEX. POMHEX treatment markedly suppresses tumor growth and reduces H4K5la and Ki67 levels in vivo . Scale bars: 250 μm. Student’s t test was used for tumor volumes (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n.s., not significant). (J) Representative IHC staining for ENO2 in human HCC (strong vs. weak) and adjacent non-tumor liver tissues. Scale bars: 250 μm. (K) Kaplan–Meier curves for OS and DFS of 102 patients with HCC grouped by ENO2 expression levels. High ENO2 expression was significantly associated with poor OS and DFS, and p values were calculated using the log-rank test. DFS, disease-free survival; H4K5ac, histone H4 lysine 5 acetylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; i.p., intraperitoneal; IHC, immunohistochemistry; KD, knockdown; OE, overexpression; OS, overall survival; shNC, negative control short-hairpin RNA; shRNA, short-hairpin RNA; shENO2, short-hairpin RNA targeting ENO2.

Article Snippet: Three human HCC cell lines, HLF (RRID: CVCL_2947), PLC/PRF/5 (RRID: CVCL_0485), and SNU475 (RRID: CVCL_0497), were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Expressing, Negative Control, Injection, Control, Immunohistochemistry, Knockdown, Over Expression, shRNA

Journal: Cell Reports Medicine

Article Title: Histone methyl-transferase G9a inhibition boosts the efficacy of immune checkpoint inhibitors in experimental hepatocellular carcinoma

doi: 10.1016/j.xcrm.2026.102717

Figure Lengend Snippet: Antitumoral effects of the G9a-specific inhibitor EZM8266 (A) Colony formation assays performed in PLC/PRF/5 human HCC cells treated with the indicated concentrations of EZM8266 ( n = 3). (B) Anchorage-independent growth assay performed in PLC/PRF/5 cells treated with the indicated concentrations of EZM8266. (C and D) Cell migration (C) and cell invasion (D) assays performed in HuH7 cells treated with the indicated concentrations of EZM8266 (48 h pretreatment and 24 h of transwell migration/invasion) ( n = 3). (E) Volcano plot representing all the genes with significant differential expression ( p < 0.05) in PLC/PRF/5 HCC cells treated with EZM8266 (5 μM for 48 h) vs . control cells and most relevant categories of differentially expressed genes identified by Gene Ontology Biological Process (GO-BP) functional classification. (F) GSEA analysis of specific categories including heatmaps with a list of genes modulated by EZM8266 selected from the RNA-seq data. (G) Effect of EZM8266, IFN-γ, and their combination on the expression of selected genes in the murine HCC cell line PM299L ( n = 3). (H) Validation of the effects of EZM8266, IFN-γ, and their combination, as indicated above, on the expression of selected genes identified in the RNA-seq analyses in the murine HCC cell line PM299L ( n = 3). (I) Experimental protocol for the study of the antitumoral effects of EZM8266 in an orthotopic model of HCC. Representative images of tumors and tumor burden (liver index) in vehicle and EZM8266-treated mice ( n = 9/group). (J) Representative images showing the immunohistochemical detection of CD4 + and CD8 + T cells and quantification of tumor-infiltrating CD8 + and CD4 + T cells (yellow arrows) in vehicle- and EZM8266-treated mice. Scale bars, 100 μm, Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 . All the replicates represent biological replicates.

Article Snippet: Human: PLC/PRF/5 , ATCC , Cat# CRL-8024 TM.

Techniques: Growth Assay, Migration, Quantitative Proteomics, Control, Functional Assay, RNA Sequencing, Expressing, Biomarker Discovery, Immunohistochemical staining