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

Journal: Histochemistry and Cell Biology

Article Title: EBF1 is expressed in pericytes and contributes to pericyte cell commitment

doi: 10.1007/s00418-021-02015-7

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: Rabbit polyclonal anti-EBF1 (WB) , MyBioSource , MBS2027769.

Techniques:

Journal: Neuroscience Bulletin

Article Title: Homeobox Gene Six3 is Required for the Differentiation of D2-Type Medium Spiny Neurons

doi: 10.1007/s12264-021-00698-5

Figure Lengend Snippet: Neurogenesis is reduced in the LGE of Six3 -cKO mice. A Upper panels. FOXP1 immunofluorescence and Ebf1 and Isl1 in situ hybridization in the LGE of control and Six3- cKO mice at E16.5. The LGE SVZ of Six3- cKO mice contains fewer FOXP1 + , Ebf1 + and Isl1 + cells than those of controls. The dotted lines indicate the border of the LGE SVZ and MZ. Lower panels, quantification of FOXP1, Ebf1 , and Isl1 . Data shown are the mean + SEM ( n = 3–4; * P < 0.05, *** P < 0.001, Student’s t -test). B Upper panels, BCL11B and EBF1 immunofluorescence in the striatum of control and Six3- cKO mice at P0. BCL11B + /EBF1 + cells represent D1 MSNs, and BCL11B + /EBF1 − cells represent D2 MSNs. Inserts show magnified images of BCL11B and EBF1 co-expression in control and Six3- cKO mice. BCL11B + /EBF1 − cells (D2 MSNs) are indicated by white dots in the right panel. Lower left panel, quantification showing that the number of BCL11B + /EBF1 − cells, but not BCL11B + /EBF1 + cells, was significantly lower in the striatum of Six3- cKO mice than in control. Lower right panel, percentages of (BCL11B + /EBF1 + )/BCL11B + and (BCL11B + /EBF1 − )/BCL11B + cells. Dotted line indicates the striatal border. Data shown are the mean + SEM ( n = 3; * P < 0.05, Student’s t -test; scale bar, 200 μm).

Article Snippet: Immunofluorescence labeling was performed with the following primary antibodies: rat anti-BCL11B (Abcam, Ab18465), rat anti-BrdU (Accurate Chemical, OBT0030s), chicken anti-β-gal (Abcam, ab9361), rabbit anti-cleaved Caspase-3 (Cell Signaling, #9661), rabbit anti-CRE (Millipore, 69050-3), rabbit anti-EBF1 (Merck, AB10523), rabbit anti-FOXP1 (Abcam, Ab16645), rabbit anti-KI67 (Abcam, ab15580), mouse anti-SIX3 (Santa Cruz Biotechnology, sc-398797), goat anti-SP8 (Santa Cruz Biotechnology, sc-104661), rabbit anti-SP9 [ ].

Techniques: Immunofluorescence, In Situ Hybridization, Expressing

Journal: Neuroscience Bulletin

Article Title: Homeobox Gene Six3 is Required for the Differentiation of D2-Type Medium Spiny Neurons

doi: 10.1007/s12264-021-00698-5

Figure Lengend Snippet: Differentiation of striatal D2 MSNs is blocked in Six3 -cKO mice. A Left panels, immunofluorescence images showing SP8 (E14.5 and E16.5) and SP9 (E16.5) expression in the LGE. Arrows indicate that SP8 + and SP9 + cells are accumulated in the vLGE SVZ of Six3- cKO mice compared to controls. Right panel, quantification data is shown ( n = 3–4). B Left panels, immunohistochemistry images showing SIX3 protein and Six3OS mRNA expression in the LGE of wild-type mice at E16.5. The magnified image shows that most, if not all, of the Six3OS + cells in the LGE SVZ express the SIX3 protein. Right panels, quantification. C Left panels, in situ hybridization for Six3OS in the LGE of control and Six3- cKO mice at E16.5 and P0. Six3OS is mainly expressed in the LGE SVZ, but the expression of Six3OS is greatly increased in the LGE of Six3- cKO mice. Note that many Six3OS + cells are distributed in the LGE MZ and striatum of Six3- cKO mice. Right panel, quantification data ( n = 3–4). D Left panels, in situ hybridization for Drd2 and Adora2a in control and Six3- cKO mice at E16.5 and P0. Drd2 and Adora2a are strongly expressed in the controls at both E16.5 and P0. However, very little Drd2 and Adora2a mRNA is expressed in the LGE and striatum of Six3- cKO mice at E16.5 and P0. Right panel, quantification data ( n = 3; mean + SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test). Note that many SP9 + cells, BCL11B + /EBF1 − cells, and Six3OS + cells are located in the LGE MZ and striatum of Six3- cKO mice in A and B . Dotted lines indicate the borders of the LGE. Scale bars, 100 μm in A , C , and D ; 50 μm in B .

Article Snippet: Immunofluorescence labeling was performed with the following primary antibodies: rat anti-BCL11B (Abcam, Ab18465), rat anti-BrdU (Accurate Chemical, OBT0030s), chicken anti-β-gal (Abcam, ab9361), rabbit anti-cleaved Caspase-3 (Cell Signaling, #9661), rabbit anti-CRE (Millipore, 69050-3), rabbit anti-EBF1 (Merck, AB10523), rabbit anti-FOXP1 (Abcam, Ab16645), rabbit anti-KI67 (Abcam, ab15580), mouse anti-SIX3 (Santa Cruz Biotechnology, sc-398797), goat anti-SP8 (Santa Cruz Biotechnology, sc-104661), rabbit anti-SP9 [ ].

Techniques: Immunofluorescence, Expressing, Immunohistochemistry, In Situ Hybridization

Journal: Histochemistry and Cell Biology

Article Title: EBF1 is expressed in pericytes and contributes to pericyte cell commitment

doi: 10.1007/s00418-021-02015-7

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: Rabbit polyclonal anti-EBF1 (IHC) , Merck Millipore , AB10523.

Techniques: