Journal: PLoS ONE

Article Title: Evidence for a functional interaction of WNT10A and EBF1 in male-pattern baldness

doi: 10.1371/journal.pone.0256846

Figure Lengend Snippet: (A) Part of the HaploReg result for rs7349332. (B) The putative WNT10A promoter element (1,641 bp) is located 434 bp upstream of the WNT10A start codon. Rs7349332, located in the third intron of WNT10A , is in high linkage disequilibrium (LD, r 2 = 0.96) with rs3856551 located in the first intron of WNT10A and within an annotated EBF1 binding site (BS). Computational in silico analyses predicted a motif similarity score (MSS) of 66% for the MPB non-risk allele (rs3856551-C) and 71% for the risk allele rs3856551-T, suggesting higher binding affinity of EBF1 with the risk allele.

Article Snippet: For the gene expression assay 0.5 μl cDNA, 5 μl TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), 0.5 μl TaqMan® Gene Expression Assay ( WNT10A : Hs00228741_m1, EBF1 : Hs01092694_m1, GAPDH : Hs02786624_g1, ACTB : Hs01060665_g1, all Applied Biosystems, Foster City, CA, USA) were mixed.

Techniques: Binding Assay, In Silico

Journal: PLoS ONE

Article Title: Evidence for a functional interaction of WNT10A and EBF1 in male-pattern baldness

doi: 10.1371/journal.pone.0256846

Figure Lengend Snippet: HEK-293T cells were co-transfected with the experimental reporters pWNT10A-FL-EBF1[C] (coral bars, non-risk allele-C) and pWNT10A-FL-EBF1[T] (blue bars, risk allele-T), respectively, and different concentrations of the EBF1 expression vector (pCMV-EBF1, EBF1). Five biological replicates for each construct were performed and WNT10A promoter activity was measured. This figure is representative for one of the five experiments in HEK-293T cells. The results for the remaining four independent experiments are shown in . The data represent the mean values ± SEM measured in the triplicate for each construct and experiment. P value was calculated with ANOVA and Tukey test. BS—binding site, FL— Firefly , LU—luminescence units.

Article Snippet: For the gene expression assay 0.5 μl cDNA, 5 μl TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), 0.5 μl TaqMan® Gene Expression Assay ( WNT10A : Hs00228741_m1, EBF1 : Hs01092694_m1, GAPDH : Hs02786624_g1, ACTB : Hs01060665_g1, all Applied Biosystems, Foster City, CA, USA) were mixed.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Activity Assay, Binding Assay

Journal: PLoS ONE

Article Title: Evidence for a functional interaction of WNT10A and EBF1 in male-pattern baldness

doi: 10.1371/journal.pone.0256846

Figure Lengend Snippet: The bar chart shows the expression values for WNT10A and EBF1 in anagen and catagen hair follicles from four independent donors (12 hair follicles each). Normalization of the WNT10A and EBF1 expression values were obtained using the measured expression levels for the housekeeping genes ACTB and GAPDH . The normalized expression values are shown relative to the gene expression in anagen hair follicles. The data show the mean values of three measurements ± SEM.

Article Snippet: For the gene expression assay 0.5 μl cDNA, 5 μl TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), 0.5 μl TaqMan® Gene Expression Assay ( WNT10A : Hs00228741_m1, EBF1 : Hs01092694_m1, GAPDH : Hs02786624_g1, ACTB : Hs01060665_g1, all Applied Biosystems, Foster City, CA, USA) were mixed.

Techniques: Expressing, Gene Expression

Journal: PLoS ONE

Article Title: Evidence for a functional interaction of WNT10A and EBF1 in male-pattern baldness

doi: 10.1371/journal.pone.0256846

Figure Lengend Snippet: Representative images of WNT10A and EBF1 protein expression in microdissected human scalp hair follicles from three different donors (independent experiments). (A) Cytosolic expression of WNT10A (green, Alexa Fluor 488) in outer and inner root sheath and hair shaft keratinocytes. (B) Nucleic expression of EBF1 (green, Alexa FLuor 488) in the dermal papilla, hair matrix, hair shaft as well as outer and inner root sheath keratinocytes. (C) Expression of WNT10A and EBF1 in the hair follicle bulge area. Nuclear counterstaining for all images was performed with DAPI (blue). (D) Illustration of EBF1 (yellow) and WNT10A (pink) expression and co-localization (orange) in microdissected human anagen hair follicles. additionally provides a higher magnification. CTS—connective tissue sheath, DAPI—4′,6-diamidino-2-phenylindole, DP—dermal papilla, HM—hair matrix, HS—hair shaft, IRS—inner root sheath, ORS—outer root sheath. Original magnification ×200.

Article Snippet: For the gene expression assay 0.5 μl cDNA, 5 μl TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), 0.5 μl TaqMan® Gene Expression Assay ( WNT10A : Hs00228741_m1, EBF1 : Hs01092694_m1, GAPDH : Hs02786624_g1, ACTB : Hs01060665_g1, all Applied Biosystems, Foster City, CA, USA) were mixed.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Evidence for a functional interaction of WNT10A and EBF1 in male-pattern baldness

doi: 10.1371/journal.pone.0256846

Figure Lengend Snippet: Based on previous research that confirmed a role of WNT signaling in the initiation and maintenance of the hair growth phase (anagen) we suggest the following functional mechanism: EBF1 binds to its recognition sequence within the WNT10A gene (2q35) and activates WNT10A expression, thereby contributing to healthy hair cycling. The binding affinity of EBF1 to its target site at 2q35 is influenced by the allelic expression of the MPB associated variant rs3856551. In the presence of the MPB risk allele (rs3856551-T), there is a decreased binding affinity of EBF1 to its target sequence. This results in a reduced expression of WNT10A as compared to carriers of the non-risk allele (rs3856551-C) that eventually leads to the MPB typical changes in the initiation and maintenance of the anagen phase.

Article Snippet: For the gene expression assay 0.5 μl cDNA, 5 μl TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), 0.5 μl TaqMan® Gene Expression Assay ( WNT10A : Hs00228741_m1, EBF1 : Hs01092694_m1, GAPDH : Hs02786624_g1, ACTB : Hs01060665_g1, all Applied Biosystems, Foster City, CA, USA) were mixed.

Techniques: Functional Assay, Sequencing, Expressing, Binding Assay, Variant Assay

Journal: PLoS ONE

Article Title: Evidence for a functional interaction of WNT10A and EBF1 in male-pattern baldness

doi: 10.1371/journal.pone.0256846

Figure Lengend Snippet: The WNT10A promoter (1,641 bp) was cloned into pGL-3-basic using Xho I and Hind III. Using In-Fusion® HD-cloning the EBF1 binding site (EBF1-BS) (93 bp) containing the rs3856551 C- or T-allele was inserted between the Bam HI and Sal I restriction sites of pGL-3-basic.

Article Snippet: For the gene expression assay 0.5 μl cDNA, 5 μl TaqMan® Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA), 0.5 μl TaqMan® Gene Expression Assay ( WNT10A : Hs00228741_m1, EBF1 : Hs01092694_m1, GAPDH : Hs02786624_g1, ACTB : Hs01060665_g1, all Applied Biosystems, Foster City, CA, USA) were mixed.

Techniques: Clone Assay, Cloning, Binding Assay

Journal: bioRxiv

Article Title: A single cell atlas of human and mouse white adipose tissue

doi: 10.1101/2021.11.09.466968

Figure Lengend Snippet: a, UMAP projection of clusters formed by 25,871 human white adipocytes. b, Expression of adipocyte marker ADIPOQ as well as specific marker genes for each adipocyte subpopulation. c, IHC for marker genes of adipocyte subpopulations hAd4, hAd5, hAd6, and hAd7 in human adipose tissue and quantification of percentage of positive adipocytes per slide in lean and obese individuals (GRIA4: 5 lean, 5 obese, 2 slides per person; PGAP1: 5 lean SAT, 4 obese SAT, 3 lean VAT, 4 obese VAT, 1 slide per person; EBF2: 3 lean, 4 obese, 2 slides per person; AGMO: 4 lean, 4 obese, 2 slides per person). Scale bars are 25 μm for GRIA4, EBF2, and AGMO, 20 μm for PGAP1. d, Estimated proportions of adipocyte subpopulations in bulk RNA sequencing data of enzymatically isolated subcutaneous adipocytes from 43 individuals plotted against subject BMI. e, Representative images of ex vivo differentiated human subcutaneous adipocytes predicted to have a low or high amount of hAd3 cells based on deconvolution of bulk RNA sequencing data. Green represents BODIPY staining, blue represents Hoechst staining. Scale bars are 100 μm. f, Normalized count of BODIPY-related features in human subcutaneous and visceral adipocytes differentiated ex vivo and stratified into low and high hAd3-containing populations. g, UMAP projection of clusters formed by 39,934 mouse white adipocytes. h, Expression of adipocyte marker Adipoq as well as specific marker genes for each mouse adipocyte subpopulation. For bar graphs, error bars represent standard error of the mean (SEM), *, p < 0.5, **, p < 0.1. For lines of best fit: hAd1 R 2 = 0.046, hAd3 R 2 = 0.0045, hAd4 R 2 = 0.043, hAd5 R 2 = 0.22, hAd1 R 2 = 0.027.

Article Snippet: The following primary antibodies and respective dilution were used: GRIA4, 1:200, Cat #23350-1-AP, Proteintech; PGAP1, 1:400, Cat. #55392-1-AP, Proteintech EBF2, 1:1000, Cat. #AF7006, R&D systems; AGMO (TMEM195) 1:100, Cat #orb395684, Biorbyt.

Techniques: Expressing, Marker, RNA Sequencing Assay, Isolation, Ex Vivo, Staining

Journal: bioRxiv

Article Title: A single cell atlas of human and mouse white adipose tissue

doi: 10.1101/2021.11.09.466968

Figure Lengend Snippet: a, Regional visualization of associations of common genetic variants near EBF2 with VATadj. b, Association of rs4872393 with VATadj, ASATadj, GFATadj, and BMI per minor allele A; n = 37,641. c, VATadj raw data plotted according to rs4872393 carrier status; n = 36,185. d, Scatterplot showing the relationship between estimated cell type proportion and the LipocyteProfiler calculated feature Mitochondrial Intensity in visceral samples. e, Expression of mitochondrial and thermogenic genes in visceral in vitro differentiated adipocytes stratified by estimated hAd6 proportion and matched for amount of differentiation using PPARG levels. f, Representative images of hAd6 low and high visceral in vitro differentiated cultures. Green represents BODIPY staining, red represents MitoTracker staining, and blue represents Hoechst staining. g, Violin plot of sNuc-seq data showing axon guidance genes in adipocyte subclusters. h, Scatterplots showing the relationship between calculated proportion of visceral subpopulations hAd2 and hAd6 and expression of pan-neuronal markers on the ambient RNA of individual visceral sNuc-seq samples. For bar graph, error bars represent SEM, *, p < .05, **, p < .01.

Article Snippet: The following primary antibodies and respective dilution were used: GRIA4, 1:200, Cat #23350-1-AP, Proteintech; PGAP1, 1:400, Cat. #55392-1-AP, Proteintech EBF2, 1:1000, Cat. #AF7006, R&D systems; AGMO (TMEM195) 1:100, Cat #orb395684, Biorbyt.

Techniques: Expressing, In Vitro, Staining

Journal: Clinical and Translational Medicine

Article Title: Long noncoding RNA IL6‐AS1 is highly expressed in chronic obstructive pulmonary disease and is associated with interleukin 6 by targeting miR‐149‐5p and early B‐cell factor 1

doi: 10.1002/ctm2.479

Figure Lengend Snippet: IL6‐AS1 interacts with EBF1 to regulate IL‐6 expression by affecting the binding of EBF1 to the IL‐6 promoter. (A) RNA pull‐down assay using IL6‐AS1 sense and antisense RNAs in HBF cells, followed by silver staining. Black arrow indicates EBF1. The interaction between IL6‐AS1 and EBF1 was confirmed by Western blotting with the extract of the RNA pull‐down assay. ILF2 and GPR94 were also detected. (B) RIP‐qPCR analysis using an anti‐EBF1 antibody showed that IL6‐AS1 interacts with endogenous EBF1 in HBF and HFL1 cells. U1 was used as the negative control (two‐way ANOVA, n = 3 biological replicates). (C) RIP‐qPCR analysis with an anti‐EBF1 antibody was conducted after overexpression of IL6‐AS1 in HBF and HFL1 cells (two‐way ANOVA, n = 3 biological replicates). (D) Schematic representation of potential EBF1 binding sites on the IL‐6 promoter. As promoters considered to be located 2000 bp upstream of the transcription start site (TSS), we designed six pairs of chromatin immunoprecipitation (ChIP)‐qPCR primers to cover the IL‐6 promoter region, with the fifth and sixth primers containing two EBF1 binding sites. (E) ChIP‐PCR analysis of EBF1 occupancy on the IL‐6 promoter in HBF and HLF cells. IgG was used as the negative control (two‐way ANOVA, n = 3 biological replicates). (F) ChIP‐PCR analysis of EBF1 occupancy on the IL‐6 promoter after transfection with an IL6‐AS1 silencer (SSIL6‐AS1) in HBF and HLF cells (two‐way ANOVA, n = 3 biological replicates). (G) Luciferase activity in the IL‐6 promoter following cotransfection of the IL6‐AS1 silencer (SSIL6‐AS1) or IL6‐AS1 overexpression vector (two‐way ANOVA, n = 4 biological replicates). (H) Luciferase activity in the IL‐6 promoter following cotransfection of an EBF1 small interfering RNA (siRNA) (siEBF1‐1) and EBF1 overexpression vector (two‐way ANOVA, n = 4 biological replicates). (I) Schematic representation of the two mutation sequences of potential EBF1 binding sites on the IL‐6 promoter. (J) Luciferase activity in the IL‐6 promoter following transfection with a reporter containing wild‐type or mutant IL‐6 promoter (one‐way ANOVA, n = 4 biological replicates). (K and L) qRT‐PCR and ELISA analysis of IL‐6 expression after transfection with two EBF1 siRNAs in HBF cells (K) or HLF cells (L) (one‐way ANOVA, n = 4 biological replicates). (M and N) qRT‐PCR and ELISA analysis of IL‐6 expression after overexpression of EBF1 in HBF cells (M) or HLF cells (N) (one‐way ANOVA, n = 4 biological replicates). (O and P) Expression of IL‐6 in HBF cells following cotransfection with IL6‐AS1 overexpression vector and EBF1 siRNA (SiEBF1‐1), determined by qRT‐PCR (O) and ELISA (P) (one‐way ANOVA, n = 5 biological replicates). (Q and R) Expression of IL‐6 in HBF cells following cotransfection with an IL6‐AS1 Smart Silencer (SSIL6‐AS1) and EBF1 overexpression vector, determined by qRT‐PCR (Q) and ELISA (R) (one‐way ANOVA, n = 5 biological replicates). Error bars represent means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: The cells were then incubated with rabbit anti‐human monoclonal antibody against EBF1 (1:200, ab108369, Abcam) overnight at 4°C, followed by incubation with Alexa Fluor 488 goat anti‐rabbit IgG (H+L) (1:500, Molecular Probes, Invitrogen) for 60 min.

Techniques: Expressing, Binding Assay, Pull Down Assay, Silver Staining, Western Blot, Negative Control, Over Expression, Chromatin Immunoprecipitation, Transfection, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Small Interfering RNA, Mutagenesis, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Translational Medicine

Article Title: Long noncoding RNA IL6‐AS1 is highly expressed in chronic obstructive pulmonary disease and is associated with interleukin 6 by targeting miR‐149‐5p and early B‐cell factor 1

doi: 10.1002/ctm2.479

Figure Lengend Snippet: Two hairpin structures of IL6‐AS1 can bind to the EBF1 protein. (A) Two computational methods (centroid plain structure and minimum free energy plain structure) were used to compute secondary structures of IL6‐AS1 using the RNAfold database ( http://rna.tbi.univie.ac.at/cgi‐bin/RNAWebSuite/RNAfold.cgi ). The sequence regions in red are the stable regions in the IL6‐AS1 secondary structure and the blue sequence regions are the unstable regions. The hairpin structures in the red and blue boxes are the potential binding sites of the EBF1 transcription factor. The structures in the black boxes are the highly conserved stem‐loops. (B) RIP‐qPCR analysis with an anti‐EBF1 antibody after transfection with wild‐type or truncated IL6‐AS1 (49‐332aa, 333‐568aa, 694‐1266aa) in HBF cells (two‐way ANOVA, n = 3 biological replicates). (C and D) Schematic representation of the EBF1 binding motif (C) and the two mutation sequences of potential EBF1 binding sites (D) in IL6‐AS1. (E and F) RIP‐qPCR analysis with an anti‐EBF1 antibody after transfection with wild‐type or mutant IL6‐AS1 in HBF cells (E) and HFL1 cells (F) (two‐way ANOVA, n = 4 biological replicates). (G and H) qRT‐PCR (G) and ELISA (H) analysis of IL‐6 expression after transfection with wild‐type or mutant IL6‐AS1 in HBF cells and HFL1 cells (one‐way ANOVA, n = 3 biological replicates). (I and J) HBF cells were cotransfected with wild‐type or mutant IL6‐AS1 and SSIL6‐AS1 and IL‐6 expression was determined by qRT‐PCR (I) and ELISA (J) (one‐way ANOVA, n = 4 biological replicates). (K and L) HBF cells were cotransfected with wild‐type or mutant IL6‐AS1 and siEBF1‐1 and IL‐6 expression was determined by qRT‐PCR (K) and ELISA (L) (one‐way ANOVA, n = 4 biological replicates). Error bars represent means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: The cells were then incubated with rabbit anti‐human monoclonal antibody against EBF1 (1:200, ab108369, Abcam) overnight at 4°C, followed by incubation with Alexa Fluor 488 goat anti‐rabbit IgG (H+L) (1:500, Molecular Probes, Invitrogen) for 60 min.

Techniques: Sequencing, Binding Assay, Transfection, Mutagenesis, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: Long noncoding RNA IL6‐AS1 is highly expressed in chronic obstructive pulmonary disease and is associated with interleukin 6 by targeting miR‐149‐5p and early B‐cell factor 1

doi: 10.1002/ctm2.479

Figure Lengend Snippet: IL6‐AS1 promotes the modification of the histones H3K4me3 and H3K27ac on the interleukin (IL) 6 promoter, probably through EBF1. (A) Schematic representation of the putative modification markers, H3K27ac, H3K4me1, and H3K4me3 upstream of IL‐6 from the ENCODE database ( https://genome.ucsc.edu/ENCODE/ ). (B–D) ChIP‐PCR analysis of H3K27ac (B), H3K4me1 (C), and H3K4me3 (D) on the IL‐6 promoter in HBF cells (two‐way ANOVA, n = 3 biological replicates). (E–H) HBF cells were transfected with SSIL6‐AS1 and assessed for H3K27ac (E), H3K4me3 (F), H3K4me1 (G) and RNA polymerase II (H) on the IL‐6 promoter by ChIP‐qPCR analysis (two‐way ANOVA, n = 3 biological replicates). (I–L) HBF cells were transfected with siEBF1‐1 and assessed for H3K27ac (I), H3K4me1 (J), H3K4me3 (K), and RNA polymerase II (L) on the IL‐6 promoter by chromatin immunoprecipitation‐qPCR analysis (two‐way ANOVA, n = 3 biological replicates). (M and N) HBF cells were cotransfected with siEBF1‐1 and IL6‐AS1 overexpression vector and assessed for H3K27ac (M) and H3K4me3 (N) on the IL‐6 promoter by ChIP‐qPCR analysis (two‐way ANOVA, n = 3 biological replicates). Error bars represent means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: The cells were then incubated with rabbit anti‐human monoclonal antibody against EBF1 (1:200, ab108369, Abcam) overnight at 4°C, followed by incubation with Alexa Fluor 488 goat anti‐rabbit IgG (H+L) (1:500, Molecular Probes, Invitrogen) for 60 min.

Techniques: Modification, Transfection, Chromatin Immunoprecipitation, Over Expression, Plasmid Preparation

Journal: Clinical and Translational Medicine

Article Title: Long noncoding RNA IL6‐AS1 is highly expressed in chronic obstructive pulmonary disease and is associated with interleukin 6 by targeting miR‐149‐5p and early B‐cell factor 1

doi: 10.1002/ctm2.479

Figure Lengend Snippet: Two mechanisms that synergize to promote interleukin (IL) 6 expression. (A and B) qRT‐PCR analysis of IL‐6 expression after cotransfection of HBF cells (A) and HFL1 cells (B) with wild‐type IL6‐AS1 or EBF1‐mutant IL6‐AS1 and miRNA‐mutant IL6‐AS1 (one‐way ANOVA, n = 5 biological replicates). (C and D) ELISA analysis of IL‐6 secretion after cotransfection of HBF cells (C) and HFL1 cells (D) with wild‐type IL6‐AS1 or EBF1‐mutant IL6‐AS1 and miRNA‐mutant IL6‐AS1 (one‐way ANOVA, n = 5 biological replicates). (E) HBF and HFL1 cells were exposed to LPS (500 ng/ml), cigarette smoke extract (CSE, 0.015%), PM 2.5 (2 μg/ml), IL17A (200 ng/ml), or nicotine (10 μM) for 24 h. qRT‐PCR analysis of IL6‐AS1 expression (two‐way ANOVA, n = 3 biological replicates). (F and G) qRT‐PCR and ELISA analysis of IL‐6 expression in wild‐type IL6‐AS1 cells, EBF1‐mutant IL6‐AS1 or miRNA‐mutant IL6‐AS1 cells after exposure to lipopolysaccharide (500 ng/ml) for 24 h in HFL1 (F) and HBF cells (G) (two‐way ANOVA, n = 3 biological replicates). (H) Correlation analysis of gene expression between IL6‐AS1/IL‐6, IL6‐AS1/miR‐149‐5p, and IL6/miR‐149‐5p from RNA‐seq results. (I) Correlation analysis between the expression of IL6‐AS1 and FEV1% in verified samples; correlation analysis between the expression of IL6‐AS1 and GOLD stage in verified samples. (J) Correlation analysis of gene expression between IL6‐AS1/IL‐6 in GSE38974 and GSE76925. (K) Correlation analysis between the expression of IL6‐AS1 and GOLD stage in GSE38974 and GSE76925. (L) Overview of the involvement of IL6‐AS1 in chronic obstructive pulmonary disease (COPD). Schematic representation of the mechanisms by which IL6‐AS1 regulates IL‐6 expression: promoting transcription and affecting histone modification by direct binding with EBF1 in the nucleus, and stabilizing IL‐6 mRNA by acting as a competing endogenous RNA (ceRNA) for miR‐149‐5p in the cytoplasm. Error bars represent the mean ± SD. * p < 0.05 and ** p < 0.01

Article Snippet: The cells were then incubated with rabbit anti‐human monoclonal antibody against EBF1 (1:200, ab108369, Abcam) overnight at 4°C, followed by incubation with Alexa Fluor 488 goat anti‐rabbit IgG (H+L) (1:500, Molecular Probes, Invitrogen) for 60 min.

Techniques: Expressing, Quantitative RT-PCR, Cotransfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Modification, Binding 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: 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: Generation and Characterization of a Knockout Mouse of an Enhancer of EBF3

doi: 10.1101/2025.01.09.631762

Figure Lengend Snippet: Ebf3 regulatory landscape and associated hs737/Rr169617 enhancer deletion mouse lines. Genome browser view of the topologically associating domain region containing Rr169617 and its target gene Ebf3 (GRCm38/mm10). The first track shows the two independent founder mouse lines generated in this study: line 299 (C57BL/6J-Rr169617 em1Tnt /J) and line 304 (C57BL/6J-Rr169617 em2Tnt /J). The second track shows the location of the regulatory region, Rr169617. The third track shows the location of human VISTA enhancers lifted over to the mouse genome. Included is hs737 that resides within the Rr169617 region. The fourth track shows enhancer-promoter interactions of Rr169617 and Ebf3 from Chen et al. 2024, Nature Genetics . The fifth track shows the genes within the region. The fifth track shows human topologically associating domains lifted over to this region and show high conservation. Finally, the chromatin state data available from ENCODE3 is shown across the different timepoints in mouse development.

Article Snippet: Taqman mouse Ebf3 (Mm00438642_m1) and GAPDH (Mm99999915_g1) gene expression assays were performed on a QuantStudio 6 Flex quantitative thermocycler using four reactions for each sample.

Techniques: Generated

Journal: bioRxiv

Article Title: Generation and Characterization of a Knockout Mouse of an Enhancer of EBF3

doi: 10.1101/2025.01.09.631762

Figure Lengend Snippet: Methylation Status at the Ebf3 Promoter in Rr169617 +/+ and Rr169617 -/- E12.5 forebrains. Shown is the methylation status of CpG sites within the Ebf3 promoter region based on the PacBio whole-genome sequencing data. The methylation patterns look similar in both. Red = methylated CpG. Blue = unmethylated CpG.

Article Snippet: Taqman mouse Ebf3 (Mm00438642_m1) and GAPDH (Mm99999915_g1) gene expression assays were performed on a QuantStudio 6 Flex quantitative thermocycler using four reactions for each sample.

Techniques: Methylation, Sequencing

Journal: bioRxiv

Article Title: Generation and Characterization of a Knockout Mouse of an Enhancer of EBF3

doi: 10.1101/2025.01.09.631762

Figure Lengend Snippet: qRT-PCR analysis for Ebf3 expression in E12.5 forebrain. A) Results of five independent qRT-PCR for Ebf3 expression. Samples for each genotype were as follows: Rr169617 +/+ (n=12), Rr169617 +/- (n=14), and Rr169617 -/- (n=10). B) Relative fold expression aggregating data across all five independent qPCR experiments. For both A and B, relative fold expression is in comparison to the Rr169617 +/+ results.

Article Snippet: Taqman mouse Ebf3 (Mm00438642_m1) and GAPDH (Mm99999915_g1) gene expression assays were performed on a QuantStudio 6 Flex quantitative thermocycler using four reactions for each sample.

Techniques: Quantitative RT-PCR, Expressing, Comparison