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: bioRxiv

Article Title: An embryonic artery-forming niche reactivates in pulmonary arterial hypertension

doi: 10.1101/2025.05.02.651303

Figure Lengend Snippet: PAH neointima features a novel population of EBF1-expressing ECs. a, Scheme of the scRNA-seq workflow. Lung endothelial (CD31 + ), stromal (Epcam - CD31 - CD45 - ), epithelial (Epcam + ), and immune (CD45 + ) populations were enriched by FACS from healthy wildtype (WT), heterozygous control ( Bmpr2 +/- mutant), or PAH ( Bmpr2 +/- mutant with intratracheal instillation of AdAlox5) rats in a previously published animal model of PAH . N=3 per animal group. The endothelial and stromal factions were submitted for scRNA-seq. b, Uniform manifold approximation and projection (UMAP) projection of annotated molecular clusters of profiled endothelial and stromal cells from all conditions. Abbreviations: Art, artery; aCap, capillary aerocyte; gCap, general capillary; EC, endothelial cell; Lym, lymphatic endothelial; ASM, airway smooth muscle; VSM, vascular smooth muscle; Peri, pericyte; AdvF, adventitial fibroblast; AlvF, alveolar fibroblast. c, Representative marker genes for each cluster visualized by dot plot, showing the fraction of expressing cells and mean expression (among expressing cells). Note novel PAH-associated cell types and their markers highlighted in red. d, Hierarchical tree showing rat lung vascular cell types and their annotated in the indicated tissue compartments. The number below the cell type name shows the total abundance of the cell type, and the stacked bar plot indicates, for each cell type, the relative abundance detected from each condition of WT, heterozygous control, or PAH rats. Black are canonical cell types per healthy reference human and mouse lung cell atlas, and red are cell types that are exclusively (100%) detected in PAH. Cell types in which a proliferative population was detected is indicated by the suffix (-p). e , Relative abundance of all the molecularly distinct cell types that were detected in PAH samples. f and g, Immunofluorescent staining of lung sections from heterozygous control or PAH rats. Protein markers shown: smooth muscle actin (SMA) for the arterial tunica media ; CD31 for endothelium, EBF1, and nuclear antigen DAPI. Yellow dotted lines outline the vessel wall and EBF1 + cells. 3D surface renderings were computed using Imaris. Note in PAH only, EBF1 + cells localizing to the sub-endothelial (“neointimal”) layer, with co-expression of CD31 antigen, with adjacent expression of SMA. h and i, Immunofluorescent staining combined with single molecule fluorescent in situ hybridization (smFISH) of human lung sections from healthy control or PAH patients. Markers shown: EBF1 (pink, RNA), endothelial marker CLDN5 (white, RNA), smooth muscle marker ACTA2 (green, RNA), smooth muscle antigen SMA (red, protein); endothelial antigen CD31 (yellow, protein), and nuclear antigen DAPI. h’ , perivascular EBF1 + cell, likely pericyte. h’’ , intimal CLDN5 + ECs and neighboring Acta2 + VSMs. White dotted lines highlight neointimal lesions in PAH. Inset shows split channel view of an occlusive neointimal lesion with co-expression of CD31 and SMA antigens, composed of 4 EBF1 + ECs ( EBF1 + CLDN5 + ACTA2 - , noted by asterisks).

Article Snippet: Chromatin was then incubated overnight with 5 μg of anti-EBF1 antibody (Santa Cruz Biotech, Sc-137065, same one used in ENCODE project) or IgG isotype control.

Techniques: Expressing, Control, Mutagenesis, Animal Model, Marker, Staining, In Situ Hybridization

Journal: bioRxiv

Article Title: An embryonic artery-forming niche reactivates in pulmonary arterial hypertension

doi: 10.1101/2025.05.02.651303

Figure Lengend Snippet: UMAP plot overlaid by lineage graph of cells from neointimal-enriched cluster (see Extended Data Fig. 1) from rat PAH lungs with single cells colored by annotated cell type, lineage graph nodes are sub-cluster centroids with size scaled by number of cells, and the edge are scaled by interaction strength between the clusters (see methods). b, UMAP plot (as in panel a) overlaid by pseudotime progression visualizing bifurcating differentiation from gCap cells to either Dll4 + arterial-like ECs (lineage 1, ‘neo-arterialization program’) or to Ebf1 + ECs (lineage 2, ‘Ebf1 program’). Arrows are the corresponding principal curves computed by slingshot, and the cells included in each of the lineages are colored by their pseudotime value, scaled from 0-1. c-d, UMAP plot (as in panel a) showing marker gene expression in units of ln (UMIs per 10,000). e, Ebf1 gene expression plotted against developmental pseudotime for cells in the Ebf1 lineage (blue) or neo-arterialization lineage (red), same as those visualized in panel b; gray shading indicates a 95% confidence interval. f-g, Loess-regression smoothened gene expression profiles of cells in the Ebf1 lineage (blue) or neo-arterialization lineage (red) plotted against developmental pseudotime, as in panel e; differentially expression genes with statistically significant association with pseudotime, as computed by tradeseq. h - i , Chord diagrams visualizing inferred endothelial- originating signaling interactions between the molecular cell types of the rat pulmonary vasculature. The width of each connection corresponds to the relative strength of the interaction inferred by cellchat.

Article Snippet: Chromatin was then incubated overnight with 5 μg of anti-EBF1 antibody (Santa Cruz Biotech, Sc-137065, same one used in ENCODE project) or IgG isotype control.

Techniques: Marker, Gene Expression, Expressing

Journal: bioRxiv

Article Title: An embryonic artery-forming niche reactivates in pulmonary arterial hypertension

doi: 10.1101/2025.05.02.651303

Figure Lengend Snippet: E b f1 + ECs arise from Aplnr + multipotent lung plexus progenitors. a, Schematic of scRNA-seq studies of pulmonary vessels during lung morphogenesis (E11.5-E15.5). Endothelial (CD31 + ) and stromal (Epcam - CD31 - CD45 - ) compartments were enriched from C57BL/6 embryonic lungs at E11.5, E12.5, E13.5, and E15.5. Colored bar indicates corresponding mouse lung developmental stage. b, UMAP plot of annotated molecular clusters of profiled endothelial and stromal cells from all embryonic ages. Abbreviations: VSM, vascular smooth muscle; ASM, airway smooth muscle; AdvF, adventitial fibroblast. c , Representative marker genes for each cluster visualized by dot plot, showing the fraction of expressing cells and mean expression (among expressing cells). d, UMAP plot overlaid by lineage graph of cells from endothelial cells of developing mouse lungs with single cells colored by annotated cell type, lineage graph nodes are sub-cluster centroids with size scaled by number of cells, and the edge are scaled by interaction strength between the clusters (see methods). e , UMAP plot (as in panel d) overlaid by pseudotime progression visualizing bifurcating differentiation from Aplnr + plexus cells to either arterial ECs (lineage 1, ‘arterialization program’) or to Ebf1 + ECs (lineage 2, ‘Ebf1 program’). Arrows are the corresponding principal curves computed by slingshot, and the cells included in each of the lineages are colored by their pseudotime value, scaled from 0-1. f, Pseudotime analyses indicating Gja5 expression in both the Ebf1 program and arterialization program. g, Loess-regression-smoothened expression ( y axis) of the indicated genes along the pseudotime trajectories. h , Schematic of a growing lung highlighting relevant vascular structures. i - k , Whole-mount staining of TdTomato, CD31, aSMA, and E-cad in an Aplnr CreERT2 ;R26 TdT omato lineage-tracing sample at E13.5. Pulse labeling initiated by 4-hydroxytamoxifen (4-OHT) at E11.5. A schematic illustration (right) shows relevant lung vasculature structures, including the proximal PA, the L1 branch (the first branch of the PA in the left lung), and the lung plexus.

Article Snippet: Chromatin was then incubated overnight with 5 μg of anti-EBF1 antibody (Santa Cruz Biotech, Sc-137065, same one used in ENCODE project) or IgG isotype control.

Techniques: Marker, Expressing, Staining, Labeling

Journal: bioRxiv

Article Title: An embryonic artery-forming niche reactivates in pulmonary arterial hypertension

doi: 10.1101/2025.05.02.651303

Figure Lengend Snippet: E B F1 specifies a transient and mobile endothelial niche expressing rich vasculotrophic signals in PA development. a, Immunofluorescent staining for TdTomato (red) and smFISH for Ebf1 (white, RNA) in an Aplnr CreERT2 ;R26 TdT omato lineage-tracing embryo at E10.5 with pulse labeling initiated by 4-OHT at E8.5. b, Whole-mount staining of CD31 (white), TdTomato (red), and EBF1(yellow) in an Aplnr CreERT2 ;R26 TdT omato lineage-tracing embryo at E13.5 with pulse labeling initiated by 4-OHT at E11.5. c, Whole-mount staining of CD31, TdTomato, ECAD, or ERG in Ebf1 CreERT2 ;R26 TdT omato embryos at E8.5 with pulse labeling initiated by 4-OHT at E11.5. Cartoon illustration demonstrates Ebf1 Cre lineage-labeled ( Ebf1 lin ) ECs within the heart-lung region. OFT, cardiac outflow track. IFT, cardiac inflow track. d-f, Whole-mount staining of CD31, TdTomato, aSMA, ECAD, or ERG in Ebf1 CreERT2 ;R26 TdT omato lineage-tracing embryos at E13.5 with pulse labeling initiated by 4-OHT at E11.5. Relevant lung vascular structures were shown, including proximal PA, L1 branch, and plexus. Yellow arrowheads highlight ERG + Tdtomato + CD31 + Ebf1 lin ECs in the lung plexus. Pink arrowheads highlight aSMA + Tdtomato + PDGFRb + Ebf1 lin stromal cells in the proximal PA. g , Migration distance of Ebf1 lin cells in ex vivo E12.5 lung cultures with pulse labeling initiated at E11.5 (See Extended Data Video 1). Four representative cells at different locations were traced in 24hr. h, Whole-mount staining of an Ebf1 CreERT2 ;R26 TdT omato embryo at E14.5 with pulse labeling initiated by 4-OHT at E11.5. i , Quantification of Ebf1 lin ECs in the lung plexus between E12.5-E14.5. n=3 per each embryonic day. j, Pseudotime analyses (referencing ) showing Ebf1 expression in both the Ebf1 program and arterialization program. k, Loess-regression-smoothened expression ( y axis) of the indicated genes along the pseudotime trajectories. l - m , Receptor-ligand interaction analyses within endothelial clusters or between endothelial and stromal populations.

Article Snippet: Chromatin was then incubated overnight with 5 μg of anti-EBF1 antibody (Santa Cruz Biotech, Sc-137065, same one used in ENCODE project) or IgG isotype control.

Techniques: Expressing, Staining, Labeling, Migration, Ex Vivo

Journal: bioRxiv

Article Title: An embryonic artery-forming niche reactivates in pulmonary arterial hypertension

doi: 10.1101/2025.05.02.651303

Figure Lengend Snippet: E b f1 + ECs are essential for pulmonary vascular development. a, Schematic of the experimental designs for panels b-e , with gene deletion initiated by 4-OHT at E8.5 (onset of the embryonic stage of lung development). b and c, Whole-mount staining of TdTomato, CD31, and aSMA in Cdh5 CreERT2 ;R26Td;Ebf1 fl/wt and Cdh5 CreERT2 ;R26Td;Ebf1 fl/fl lungs at E13.5. Yellow dotted lines outline the PAs and their branches. d, Quantification of plexus volume and PA diameter in both genotypes. N=4 per group. e , Volcano plot displaying differential expressed genes in the plexus population, comparing Ebf1 fl/fl KOs vs Cre- littermates. Endothelial and stromal cells were enriched for scRNA-seq. n=8 per each genotype group. f , Schematic of the experimental designs for panels g-i , with gene deletion initiated by 4-OHT at E11.5 (onset of the pseudoglandular stage). g and h, Whole-mount staining of CD31, aSMA, and ECAD in Cdh5 CreERT2 ;Ebf1 fl/wt and Cdh5 CreERT2 ;Ebf1 fl/fl lungs at E15.5. Yellow dotted lines outline the distal PAs and their branches. Yellow arrowheads highlight defects in PA branching and reduced aSMA coverage in Ebf1 fl/fl KO embryos. i , Volcano plot displaying differential expressed genes in the stromal populations, comparing Ebf1 fl/fl KOs vs Cre- littermates. Endothelial and stromal cells were enriched for scRNA-seq. n=8 per each genotype group. j, Schematic of the experimental designs for panels k-m . k and l, Whole-mount staining of CD31 (white), aSMA (green), and Elastin (cyan) in adult Ebf1 lsl ;Cdh5 - ( Cre- control) and Cdh5 CreERT2 ; Ebf1 lsl lungs. Yellow dotted lines outline the distal PAs. Yellow arrowheads highlight less aSMA coverage in Cre- distal PAs. m, Quantification of aSMA coverage in distal PAs between genotype groups. N=6 per each genotype group.

Article Snippet: Chromatin was then incubated overnight with 5 μg of anti-EBF1 antibody (Santa Cruz Biotech, Sc-137065, same one used in ENCODE project) or IgG isotype control.

Techniques: Staining, Control

Journal: bioRxiv

Article Title: An embryonic artery-forming niche reactivates in pulmonary arterial hypertension

doi: 10.1101/2025.05.02.651303

Figure Lengend Snippet: T h e core artery-forming niche driven by Ebf1 + ECs. a, Proposed model of the Ebf1 + ECs-driven, artery-forming niche in PA development. During lung morphogenesis, multipotent Aplnr + plexus progenitors give rise to both pre-arterial ECs and an Ebf1 + endothelial niche population. Without directly incorporating into the PA endothelium, these Ebf1 + ECs serve as the “ PA organizing cells ”. They secrete vasculotrophic signals required for the proliferation and arterialization of the plexus progenitors, as well as for the recruitment and muscularization of the stromal progenitors, thereby controlling the angiogenic expansion, branching, and maturation of the growing PAs. b , Working model of the Ebf1 + ECs-driven, artery-forming niche in adult PAH. Under conditions of inflammation and BMPR2 deficiency, Aplnr + gCap cells re-acquire stem-like properties and differentiate into Dll4 + arterial-like ECs and Ebf1 + ECs. Through comparable vasculotrophic signaling, these Ebf1 + ECs induce the proliferation and reprogramming of gCap cells, as well as the recruitment and activation of cells from the stromal layers, thereby contributing to PA vascular remodeling.

Article Snippet: Chromatin was then incubated overnight with 5 μg of anti-EBF1 antibody (Santa Cruz Biotech, Sc-137065, same one used in ENCODE project) or IgG isotype control.

Techniques: Activation Assay