Journal: bioRxiv

Article Title: EBF1 regulates the blood-thymus barrier and ETP influx through Claudin-5 in thymic endothelial cells

doi: 10.64898/2026.01.22.700802

Figure Lengend Snippet: (A) Normalized read counts of Ebf1 in different thymic stromal populations from public RNA-sequencing dataset [original data from ]. In this study a distinction was made between KitL + and KitL - thymic EC cells which have been represented a single endothelial cell (EC) population in this graph. Statistical significance was determined by one-way ANOVA. (B) Quantitative reverse-transcriptase (qRT) PCR of Ebf1 relative to Actb mRNA expression. Data are represented as mean ± sem. n=3. Statistical significance was determined by unpaired t-test. (C) Representative flow cytometry plots of TECs (CD45 - Ter119 - Epcam + ) and ECs (CD45 - Ter119 - CD31 + ) in Ebf1 WT and Ebf1 KO mice. (D) Frequency of TECs and thymic ECs in CD45-thymic stromal cells. Ebf1 WT n=7, Ebf1 KO n=7. Mice were analyzed at 4-6-weeks-old, (•) represent female mice, (▾) represent male mice. Data are represented as boxplots. Statistical significance was determined by unpaired t-test. Data are from ≥ 2 independent experiments. mTEC, medullary thymic epithelial cells; cTEC, cortical thymic epithelial cells; ECs, endothelial cells.

Article Snippet: Quantitative RT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific), with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: RNA Sequencing, Reverse Transcription, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: EBF1 regulates the blood-thymus barrier and ETP influx through Claudin-5 in thymic endothelial cells

doi: 10.64898/2026.01.22.700802

Figure Lengend Snippet: (A) Representative dot plots of ETPs (CD4 - CD8 - Lin - cKit + ) in Ebf1 WT and Ebf1 KO mice. (B) Total frequency (left) and absolute number (right) of ETPs in the thymus. (C) Representative contour plots for the different DN populations in Ebf1 WT and Ebf1 KO mice. (D) Absolute number of DN1-4 populations in the thymus. (B, D) Ebf1 WT n=12, Ebf1 KO n=13. (E) Total spleen cellularity. Ebf1 WT n=6, Ebf1 KO n=5. (F) Frequency of CD4 and CD8 T cells within CD19 - cells in the spleen. Ebf1 WT , Ebf1 KO n=4. (G) Absolute number of lymphoid-biased MPP4 (Lin - Sca1 + cKit + CD150 - CD48 + Flt3 + ) progenitors in the bone marrow. Ebf1 WT n=10, Ebf1 KO n=8. (H) Absolute number of lymphoid progenitors CLP (Lin - Sca1 int cKit int IL7R + Flt3 + ) progenitors in the bone marrow. Ebf1 WT , Ebf1 KO n=6. (I) Representative dot plots of ALP and BLP populations in Ebf1 WT and Ebf1 KO mice. (J) Proportion of ALP and BLP cells in the CLP population. Ebf1 WT n=8, Ebf1 KO n=7. (A-J) Mice were analyzed at 4-6-weeks-old, (•) represent female mice, (▾) represent male mice. Data are represented as boxplots. Statistical significance was determined by unpaired t-test. Data are from ≥ 2 independent experiments. ETPs, early thymic progenitors; DN, double negative (CD4 - CD8 - Lin - cKit - ).

Article Snippet: Quantitative RT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific), with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques:

Journal: bioRxiv

Article Title: EBF1 regulates the blood-thymus barrier and ETP influx through Claudin-5 in thymic endothelial cells

doi: 10.64898/2026.01.22.700802

Figure Lengend Snippet: (A) Representative histograms of GATA3 expression in DN1-4 populations of the thymus, in Ebf1 WT and Ebf1 KO mice. (B, C) Mean fluorescence intensity (MFI) of Gata3 (B) in DN1-4 populations, and (C) in ETPs, in the thymus. Ebf1 WT n=8, Ebf1 KO n=9 . (D) Frequency of Ki67+expressing cells within ETPs. (E) Frequency of Caspase3+ expressing cells within ETPs. (D, E) Ebf1 WT n=7, Ebf1 KO n=8. (F) Representative dot plots for ETP1 and ETP2 populations in Ebf1 WT and Ebf1 KO mice. (G) Absolute number of ETP1 and ETP2 populations in the thymus. (H) Frequency of ETP1 and ETP2 cells in the ETP population. (G, H) Ebf1 WT n=12, Ebf1 KO n=13. (I) Frequency of Ki67+ expressing cells within ETP1 and ETP2 cells. Ebf1 WT n=7, Ebf1 KO n=8. Mice were analyzed at 4–6-weeks-old, (•) represent female mice, (▾) represent male mice. Data are represented as boxplots. Statistical significance was determined by unpaired t-test. Data are from ≥ 2 independent experiments.

Article Snippet: Quantitative RT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific), with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: EBF1 regulates the blood-thymus barrier and ETP influx through Claudin-5 in thymic endothelial cells

doi: 10.64898/2026.01.22.700802

Figure Lengend Snippet: (A) MA plot of thymic ECs comparing Ebf1 WT and Ebf1 KO transcriptomes. Dots in blue represent genes with an FDR <0.05. The y axis represents shrunken log 2 fold change (FC), capped at |log 2 FC| 2, and the x axis represents log10 basemean. The number of up-and down-regulated genes in Ebf1 KO mice compared to Ebf1 WT mice are displayed. (B) Cnetplot analysis of DE genes between Ebf1 WT and Ebf1 KO thymic ECs. (C) Heatmap showing gene expression of DE genes associated with gene-ontology (GO) signature: regulation of vasculature development. Heatmap scale represents z-scores. (D) Normalized read counts of Cldn5 in different thymic stromal populations from public RNA-sequencing dataset [original data from ]. (E) Normalized read counts of Cldn5 in Ebf1 WT and Ebf1 KO thymic ECs. Mice were analyzed at 4–6-weeks-old, n=3. (F) Schematic diagram illustrating reduced permeability of the blood-thymus barrier and impaired trafficking of thymic-seeding progenitors (TSP), associated with increased Claudin-5 expression in Ebf1 KO thymic ECs. EC, endothelial cells; cortical thymic epithelial cells (cTEC); medullary thymic epithelial cells (mTEC); DE, differentially expressed; FDR, false discovery rate.

Article Snippet: Quantitative RT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific), with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Gene Expression, RNA Sequencing, Permeability, 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: 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: iScience

Article Title: Serine synthesis pathway regulates cardiac differentiation from human pluripotent stem cells

doi: 10.1016/j.isci.2025.112843

Figure Lengend Snippet: scRNA-seq analysis reveals changes in cell populations under SSP inhibition (A) The UMAP plot of scRNA-seq data displays individual cells by cell types. (B) The dot plot shows key marker genes identified in each cell cluster. (C) UMAP plot showing the expression of genes indicative of mesoderm, endoderm, and ectoderm. (D) Bar plot showing the distribution of cells in each sample. (E) Bar graph showing GO enrichment analysis for the upregulated genes in Cluster 9. (F) UMAP plot showing the expression of genes indicative of CM ( TNNT2 and ACTN2 ) and CPM ( TBX1 , EBF1 , EBF2 , and TCF21 ). CF, cardiac fibroblast; CM, cardiomyocyte; CPC, cardiac progenitor cell; CPM, cardiopharyngeal mesoderm; EC, endothelial cell; ER, endoplasmic reticulum; PSC, pluripotent stem cell; scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: EBF1 (Hs01092694) , Thermo Fisher Scientific , Cat# 4331182.

Techniques: Inhibition, Marker, Expressing, RNA Sequencing