Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: ERS upregulates CLGN expression in HCC. (A) Volcano plot of differentially expressed genes from mRNA sequencing of Hep-G2 cells. Red and blue dots represent significantly up- and down-regulated genes, respectively (CLGN is labeled). (B) Heatmap of the top 25 up- and down-regulated genes from mRNA sequencing. (C) Expression levels of the top 25 upregulated genes in HCC and adjacent normal tissues from the TCGA database. (D–F) Kaplan-Meier survival analysis of HCC patients stratified by high and low expression of CLGN (D) , GPR1 (E) , and UNC5B (F) . (G) qRT–PCR analysis of candidate gene expression in Hep-G2 cells treated with or without TM (unpaired Student’s t-test). (H, I) Dose-dependent effects of the ERS inducer TM on CLGN and GRP78 expression in Hep-G2 cells, as determined by qRT–PCR (H) and Western blot (I) (one-way ANOVA with Dunnett’s post hoc test). (J) CLGN protein expression under UPR pathway inhibition. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Expressing, Sequencing, Labeling, Quantitative RT-PCR, Gene Expression, Western Blot, Inhibition

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: High CLGN expression correlates with aggressive clinicopathological features and poor prognosis in HCC. (A) CLGN mRNA expression in unpaired HCC and normal liver tissues from the TCGA-LIHC cohort (unpaired Student’s t-test). (B–F) Analysis of CLGN mRNA expression levels in the TCGA cohort stratified by (B) tumor status, (C) age, (D) sex, (E) serum AFP level, and (F) histological grade (unpaired Student’s t-test or one-way ANOVA). (G) Sankey diagram illustrating the flow and association between TNM stage, histological grade, CLGN expression level, and tumor status. (H) IHC images of CLGN staining in HCC tissues, classified into four grades (0-3) based on staining intensity. (I) Statistical analysis of CLGN IHC scores in HCC tissues compared with adjacent non-tumor tissues (paired Student’s t-test). (J–L) Analysis of CLGN IHC scores stratified by (J) hepatitis status, (K) liver cirrhosis status, and (L) tumor size (unpaired Student’s t-test). (M, N) Correlation between CLGN protein expression and the ERS markers (M) GRP78 and (N) ATF6. Patients were grouped based on the median IHC score of each ERS marker (unpaired Student’s t-test). (O) Kaplan-Meier analysis of overall survival based on CLGN IHC staining in our institutional cohort (n=35, Log-rank test). (P, Q) Kaplan-Meier survival analysis of the TCGA-LIHC cohort based on CLGN mRNA expression levels, showing (P) disease-specific survival and (Q) overall survival (Log-rank test). (R) Western blot analysis of CLGN protein expression in 8 paired fresh-frozen HCC (T) and adjacent non-tumor (N) tissues. GAPDH was used as a loading control. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Expressing, Staining, Marker, Immunohistochemistry, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN promotes HCC cell proliferation in vitro . (A, B) Proliferation of Hep-G2 cells with stable CLGN knockdown was assessed by (A) colony formation assay and (B) CCK-8 assay. (C, D) Proliferation of Huh-7 cells with stable CLGN knockdown was assessed by (C) colony formation assay and (D) CCK-8 assay. (E, F) Proliferation of Hep-3B cells with stable CLGN overexpression was assessed by (E) colony formation assay and (F) CCK-8 assay. (G) Proliferation of CLGN-knockdown Hep-G2 and Huh-7 cells was assessed by EdU assay. Scale bar, 50 μm. (H) Proliferation of CLGN-overexpressing Hep-3B cells was assessed by EdU assay. Scale bar, 50 μm. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test or one-way ANOVA).

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: In Vitro, Knockdown, Colony Assay, CCK-8 Assay, Over Expression, EdU Assay

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN promotes invasion, migration, and suppresses apoptosis in HCC cells in vitro . (A, B) Effects of CLGN knockdown in Hep-G2 cells on (A) wound healing migration and (B) Transwell invasion. (C, D) Effects of CLGN knockdown in Huh-7 cells on (C) wound healing migration and (D) Transwell invasion. (E, F) Effects of CLGN overexpression in Hep-3B cells on (E) wound healing migration and (F) Transwell invasion. (G) Apoptosis analysis by flow cytometry in CLGN-knockdown Hep-G2 and Huh-7 cells. (H) Apoptosis analysis by flow cytometry in CLGN-overexpressing Hep-3B cells. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test or one-way ANOVA).

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Migration, In Vitro, Knockdown, Over Expression, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN knockdown enhances the anti-tumor efficacy of Pae by modulating ERS. (A, B) Hep-G2 control and CLGN-knockdown cells were treated with TM and/or Pae, followed by analysis of (A) apoptosis via flow cytometry and (B) clonogenic survival. (C) Representative images of resected tumors from the xenograft mouse model under different treatment conditions. (D) Tumor weights from each treatment group at the endpoint. (E) IHC analysis of Ki67, CLGN, and NF-κB expression in xenograft tumor tissues. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (B, D: one-way ANOVA with Tukey’s post hoc test; A: two-way ANOVA).

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Knockdown, Control, Flow Cytometry, Expressing

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN suppresses apoptosis through activation of the NF-κB pathway. (A) Volcano plot of DEGs from RNA sequencing of control versus CLGN-knockdown Hep-G2 cells. (B) Chord plot illustrating the results of combined GO/KEGG and logFC enrichment analysis for the identified DEGs. (C) Bar graph of the most significantly enriched KEGG pathways. (D) Western blot analysis of key NF-κB pathway proteins in Hep-G2 with CLGN knockdown and Hep-3B cells with CLGN overexpression. (E) Western blot analysis of CLGN, NF-κB, and Bcl-2 expression in control and CLGN-knockdown Hep-G2 cells treated with TM or TM+Pae. (F) Western blot analysis of CLGN, NF-κB, and Bcl-2 expression in vector-control and CLGN-overexpressing Hep-3B cells treated with the NF-κB inhibitor PDTC or Pae. GAPDH was used as a loading control for all Western blot analyses.

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Activation Assay, RNA Sequencing, Control, Knockdown, Western Blot, Over Expression, Expressing, Plasmid Preparation