Journal: Nature Communications

Article Title: Functional genomics uncovers the transcription factor BNC2 as required for myofibroblastic activation in fibrosis

doi: 10.1038/s41467-022-33063-9

Figure Lengend Snippet: A Input and BNC2 ChIP-seq signals together with CoP-seq and H3K27ac ChIP-seq signals from LX2 cells were visualized at the BNC2 cistrome (1479 binding sites). Heatmaps at the bottom show the signals in 5 kb regions centered on the BNC2 peaks. Average signals are plotted on top of the heatmaps. B The BNC2 cistrome was used to search for proteins with similar genomic binding profiles in CistromeDB and the top 20 hits were ranked according to the GIGGLE similarity score. Proteins involved in TGFβ signaling (SMADs) and Hippo/YAP1 (TEAD and YAP1) were highlighted in red and green, respectively. C Analyses performed as in A to monitor SMAD3 and YAP1 ChIP-seq signals from MFs at the BNC2 cistrome. D Transcriptional regulators specifically identified in BNC2 RIME but not IgG control RIME were clustered according to the percent protein coverage in individual biological replicates ( n = 3 biologically independent experiments; anti-BNC2 antibody 55220-1-AP, Proteintech). Percent coverage obtained in an additional BNC2 RIME experiment using the anti-BNC2 antibody HPA018525 (Sigma-Aldrich) is shown on the right. E Nuclear extracts from LX2 cells expressing recombinant BNC2 and YAP1 were subjected to immunoprecipitation with an antibody against BNC2 (55220-1-AP, Proteintech). Immunoprecipitated material was analyzed by western blot using antibodies directed against BNC2 or YAP1. The presented data are representative of two biologically independent experiments. MW, molecular weight markers. F , G The Integrated Genome Browser (IGB) was used to visualize ChIP-seq profiles for BNC2 (black track, LX2 cells), SMAD3 (red track, LX2 cells), YAP1 (green track, IMR90 cells), and H3K27ac (blue track, MF-HSCs) at the COL1A1 ( F ) and BNC2 ( G ) genes.

Article Snippet: LX2 cells were transfected for 48 h with a plasmid coding for human BNC2 (custom construction, E-Zyvec) and a plasmid coding for constitutively active nuclear YAP1 (YAP1-S127A, Addgene).

Techniques: ChIP-sequencing, Binding Assay, Control, Expressing, Recombinant, Immunoprecipitation, Western Blot, Molecular Weight

Journal: Nature Communications

Article Title: Functional genomics uncovers the transcription factor BNC2 as required for myofibroblastic activation in fibrosis

doi: 10.1038/s41467-022-33063-9

Figure Lengend Snippet: A RT-qPCR data showing changes in gene expression upon murine primary Q-HSCs spontaneous in vitro activation into MF-HSCs ( n = 6 biologically independent experiments). Log 2 fold changes (FC) between MF-HSCs (6 days of culture) and Q-HSCs (1 day of culture) are shown. B RT-qPCR data showing changes in gene expression upon YAP1 inhibition in MF-HSCs. Isolated mouse primary HSCs were treated with 1 µM verteporfin or vehicle for 6 days before being harvested [ n = 3 ( Yap1 ), 4 ( Bnc2 and Ctgf ), or 5 ( Col1a1 and Acta2 ) biologically independent experiments]. Log 2 FC between verteporfin and vehicle-treated cells are shown. C RT-qPCR data showing changes in gene expression upon Yap1 silencing in MF-HSCs ( n = 3 biologically independent experiments). Log 2 FC between siYAP1 and siCTRL-transfected cells are shown. D RT-qPCR data showing differences in gene expression in mouse primary HSCs grown for 9 days in 3D (spheroids) or in 2D [ n = 3 ( Bnc2 ) or 4 ( Col1a1 , Acta2 , Yap1 , Ctgf ) biologically independent experiments). Log 2 FC between cells grown in 3D and 2D are shown. B – D Expression of Ctgf , an established YAP1 target, was assessed. E RT-qPCR data showing changes in gene expression induced by treatment of MF-HSCs with TGFβ (1 ng/mL) for 24 h ( n = 4 biologically independent experiments). Log 2 FC between cells treated with TGFβ and vehicle are shown. F RT-qPCR data showing changes in gene expression induced by treatment of EMS404 MF-HSCs with the indicated TGFβ signaling inhibitors for 24 h ( n = 3 biologically independent experiments). Log 2 FC between cells treated with TGFβ signaling inhibitors and vehicle are shown. In all panels, bar graphs show means ± SD. Statistical significance was assessed using two-sided one-sample t test with Benjamini–Hochberg correction for multiple testing to determine if the mean log 2 FC was statistically different from 0. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: LX2 cells were transfected for 48 h with a plasmid coding for human BNC2 (custom construction, E-Zyvec) and a plasmid coding for constitutively active nuclear YAP1 (YAP1-S127A, Addgene).

Techniques: Quantitative RT-PCR, Gene Expression, In Vitro, Activation Assay, Inhibition, Isolation, Transfection, Expressing

Journal: Communications Biology

Article Title: Exosomal miR-212-5p promotes tendon repair via targeting FOXO1 to activate PP1A/YAP1 signaling

doi: 10.1038/s42003-025-09210-5

Figure Lengend Snippet: A qPCR was performed to assess FOXO1 expression in TDSCs after treatment with exosomes derived from miR-212-5p -depleted ADSCs. B Western blot was conducted to evaluate FOXO1 and p-YAP1 levels after treatment with exosomes derived from miR-212-5p-depleted ADSCs. C Bioinformatics analysis using the Starbase database was performed to predict the target interaction between miR-212-5p and FOXO1 . D qPCR was used to measure miR-212-5p levels in TDSCs after transfection with miR-212-5p mimics. E Dual-luciferase reporter assay was used to assess luciferase activity after treatment with exosomes derived from miR-212-5p -depleted or miR-212-5p -overexpressing ADSCs. F RNA pull-down assay was conducted using biotin-labeled miR-212-5p to evaluate its binding interaction with FOXO 1. G qPCR was performed to assess FOXO 1 expression after transfection with shFOXO1 in ADSCs. H Western blot analysis was used to evaluate FOXO1 and p-YAP1 levels after knockdown of miR-212-5p in ADSC-Exos and simultaneous knockdown of FOXO1. Data are presented as mean ± SD, and statistical analysis was conducted using a student t-test (for D , F – G ) of one-way ANOVA followed by Tukey’s post hoc test (for A , B , E , H ). n = 3.*p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cDNA ORF coding full sequences for YAP1 (Origene, Shanghai, China) mutant serine-to-alanine mutation at position 127 (S127A) was generated through PCR-based site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Sacramento, CA, USA) following the manufacturer ́s protocol, and was subcloned into an overexpressing vector.

Techniques: Expressing, Derivative Assay, Western Blot, Transfection, Luciferase, Reporter Assay, Activity Assay, Pull Down Assay, Labeling, Binding Assay, Knockdown

Journal: Communications Biology

Article Title: Exosomal miR-212-5p promotes tendon repair via targeting FOXO1 to activate PP1A/YAP1 signaling

doi: 10.1038/s42003-025-09210-5

Figure Lengend Snippet: A , B qPCR and Western blot were performed to analyze PP1A levels in TDSCs transfected with shNC or shFOXO1 after treatment with exosomes derived from miR-212-5p-depleted ADSCs. C The JASPAR database was used to predict FOXO1 binding sites on the PP1A promoter. D ChIP assay was conducted using a FOXO1 antibody to assess its binding to the PP1A promoter. E , F qPCR and Western blot were performed to evaluate FOXO1 expression in TDSCs after transfection with FOXO1 overexpression vectors. G Dual-luciferase reporter assay was used to investigate the impact of FOXO1 knockdown and overexpression on PP1A transcriptional activity. H qPCR was performed to assess PP1A expression following FOXO1 knockdown and overexpression in TDSCs. I qPCR was conducted to evaluate PP1A expression after transfection with shPP1A in TDSCs. J Western blot was performed to study the effects of FOXO1 and PP1A knockdown on PP1A and p-YAP1 levels. K Immunofluorescence was used to examine nuclear YAP1 levels (green) in TDSCs after FOXO1 and PP1A knockdown. Scale bar: 100 μm. Data are presented as mean ± SD, and statistical analysis was conducted using a student t-test (for D – F , I ) of one-way ANOVA followed by Tukey’s post hoc test (for A , B , G , H , J ). n = 3 **p < 0.01, ***p < 0.001.

Article Snippet: The cDNA ORF coding full sequences for YAP1 (Origene, Shanghai, China) mutant serine-to-alanine mutation at position 127 (S127A) was generated through PCR-based site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Sacramento, CA, USA) following the manufacturer ́s protocol, and was subcloned into an overexpressing vector.

Techniques: Western Blot, Transfection, Derivative Assay, Binding Assay, Expressing, Over Expression, Luciferase, Reporter Assay, Knockdown, Activity Assay, Immunofluorescence

Journal: Communications Biology

Article Title: Exosomal miR-212-5p promotes tendon repair via targeting FOXO1 to activate PP1A/YAP1 signaling

doi: 10.1038/s42003-025-09210-5

Figure Lengend Snippet: A Western blot was performed to analyze the effects of transfection with wild-type YAP1 (YAP1-WT) expression vectors or S127A mutant YAP1 expression vectors (YAP1-S127A) on YAP1 and p-YAP1 levels. B – F MTT, EdU staining, scratch assay, Transwell assay, and Western blot were used to evaluate TDSC proliferation, migration, and the expression of tenogenic differentiation markers, including TNC, TNMD, Scx, COL1A1, and GAPDH, after FOXO1 overexpression with simultaneous overexpression of the S127A mutant YAP1. Scale bar: 100 or 500 μm. G Alcian Blue staining was performed to evaluate cartilage-like matrix deposition in TDSCs. Scale bar: 100 μm. Data are presented as mean ± SD, and statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test. n = 3. **p < 0.01, ***p < 0.001.

Article Snippet: The cDNA ORF coding full sequences for YAP1 (Origene, Shanghai, China) mutant serine-to-alanine mutation at position 127 (S127A) was generated through PCR-based site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Sacramento, CA, USA) following the manufacturer ́s protocol, and was subcloned into an overexpressing vector.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Staining, Wound Healing Assay, Transwell Assay, Migration, Over Expression

Journal: Communications Biology

Article Title: Exosomal miR-212-5p promotes tendon repair via targeting FOXO1 to activate PP1A/YAP1 signaling

doi: 10.1038/s42003-025-09210-5

Figure Lengend Snippet: A Hematoxylin and eosin (HE) staining was performed to evaluate the arrangement of fibrous tissue in tendons from mice treated with exosomes derived from normal or miR-212-5p -depleted ADSCs. Scale bar: 50 or 20 μm. B Safranin O/Fast Green staining of tendon sections to evaluate ectopic cartilage-like matrix formation after injury in different groups of mice. Scale bar: 50 or 20 μm. C Immunohistochemistry (IHC) was used to assess the levels of TNMD, Scx, COL1A1, and SOX9 in tendon tissues from different groups of mice. Scale bar: 20 μm D , E Western blot analysis was conducted to examine the expression of tenogenic differentiation markers, including TNC, TNMD, Scx, and COL1A1, as well as PP1A, FOXO1, and p-YAP1 levels in tendon tissues from different groups of mice. F , G Biomechanical testing was performed to measure the failure load and stiffness of tendon tissues from different groups of mice. Data are presented as mean ± SD, and statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test. n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cDNA ORF coding full sequences for YAP1 (Origene, Shanghai, China) mutant serine-to-alanine mutation at position 127 (S127A) was generated through PCR-based site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Sacramento, CA, USA) following the manufacturer ́s protocol, and was subcloned into an overexpressing vector.

Techniques: Staining, Derivative Assay, Immunohistochemistry, Western Blot, Expressing