Fig 1: Bcl-3 regulates adipo-osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro and protects BMSCs from senescence. (A) Bcl-3 was knockdown in BMSCs and cultured in differentiation medium. Cell differentiation was assessed by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining after14 days of osteogenic induction. Scale bar, 5 mm (above) and 200 µm (below). (B) The expression of osteogenesis markers (OCN, Osterix and Runx2) were analysed 14 days after osteogenic induction of Bcl-3 knock-out BMSCs. (N = 4 independent experiments). (C) Bcl-3 was knockdown in BMSCs and cultured in differentiation medium. Cell differentiation was assessed by oil red staining after 21 days of adipogenic induction. Scale bar, 5 mm (above) and 200 µm (below). (D) The expression of adipogenic markers (Fabp4, Adipoq and PPAR?) was analysed 6 days after adipogenic induction of Bcl-3 knock-out BMSCs. (N = 4 independent experiments). (E) Western blot detection of GAPDH, Bcl-3, Osterix and Fabp4 protein in BMSCs. The data are presented as the mean ± SD. (F) Western blot detection of GAPDH and Bcl-3 protein levels in H2O2 treated BMSCs. (G,H) Representative images and quantification of CFU-Fs formed by cells from Bcl-3–/– and wild-type (WT) mice. Scale bar, 0.5 mm. (N = 3 independent experiments). (I) SA-ß-gal staining of Bcl-3 knockdown BMSCs and quantitative analysis of SA-ß-gal staining. Scale bar, 200 µm. (N =3 independent experiments). (J) Bcl-3, P16 and P21 mRNA in Bcl-3 knockdown BMSCs was assessed by qRT-PCR. (N = 4 independent experiments). The data are presented as the mean ± SD. *P <.05; **P <.01; ***P < .005; ****P < .0001 vs. control group. Statistical analysis was performed using Student's t-test (B, D, I and J) and two-way ANOVA test (H). Primary antibodies: GAPDH, Abcam(ab181602); BCL-3, Abcam(ab259832); FABP4, Abcam(ab92501); OSTERIX, Abcam(ab209484)
Fig 2: Endogenous TG2 is robustly S-nitrosylated in Mouse Tissues. SNO-TG2 isolated from three separate mouse kidneys (designated as 1, 2 and 3). 10 µg samples were loaded on to each lane for immunoblot analysis. (A) 0.5% of total input (B). Proteins eluted from ascorbate treated samples (+ascorbate) = SNO-proteins. (C). Proteins eluted from untreated samples (-ascorbate) = controls. SNO-modified TG2 was eluted from thiopropyl-Sepharose beads and analyzed by immunoblots using mouse monoclonal antibody against TG2 (cub7402, ThermoFisher Scientific) as described under Materials and Methods. GAPDH, a prototypic SNO-modified protein (detected using rabbit monoclonal antibody Abcam, Ab181602) is included as a control.
Fig 3: Lower expression of JUN and FOS and positive correlation to RXFP1 in control and IPF lungs and lung fibroblasts.Lung tissue expression levels of FOS analyzed using microarray (A) and JUN analyzed using bulk RNA sequencing (C) from the publicly available Lung Genomics Research Consortium (LGRC) gene expression dataset (GEO accession GSE47460; http://www.lung-genomics.org/) were compared between control (108 subjects for FOS and 22 subjects for JUN) and idiopathic pulmonary fibrosis (IPF) (160 subjects for FOS and 22 subjects for JUN). The mean and standard deviation for each group and Mann-Whitney U test p-values are shown. Correlation of FOS (B) and JUN (D) gene expression levels with RXFP1 was analyzed in IPF lungs (160 subjects for FOS and 22 subjects for JUN) using linear regression and the R2 and p-value are shown. (E) Protein levels of JUN and FOS in independent IPF and control lung fibroblast lines analyzed by western blot with antibodies specific for JUN (rabbit mAb, Cell signaling technology #9165) and FOS (mouse mAb, Protein tech # 66590-1-IG). GAPDH (rabbit mAb Abcam Abcam ab181602) was used as a sample loading control. Total proteins isolated from confluent fibroblasts using radioimmunoprecipitation assay (RIPA) were separated by SDS-PAGE gel electrophoresis. (F) Densitometry of the results on (E) using the ImageJ software (National Institutes of Health) [29]. For FOS, total density of both bands was used for each sample. A total of 10 independent lung fibroblast lines (5 IPF and 5 control) were analyzed.
Fig 4: Bcl-3 activates Wnt/ß-catenin signalling pathway and maintains ß-catenin functions in vitro. (A,B) Gene ontology (GO, Left) and Kyoto encyclopedia of genes and genomes (KEGG, Right) analysis of downregulated genes with Bcl-3 deletion in bone marrow mesenchymal stem cells (BMSCs). (C) Heat map showed relative genes expression in Wnt pathway of shCtrl and shBcl-3 BMSCs. (D) Gene set enrichment analysis (GSEA) showed a significant decrease of Wnt/ß-catenin gene signatures in shBcl-3 BMSCs. (E) Lysates from BMSCs were used, and anti-Bcl-3 antibody was used in IP followed by immunoblot using the indicated antibodies. (F) Bcl-3, Ac-K49-ß-catenin and ß-catenin were analysed by immunoblot in control and Bcl-3-knockdown BMSCs after treated with Wnt 3a for 0 h or 4 h. (G) The levels of Bcl-3 in nuclear and cytoplasmic. (H) Bcl-3, Ac-K49-ß-catenin and ß-catenin were analysed by immunoblot in control and Bcl-3-knockdown BMSCs after treated with cycloheximide (CHX, 50 mg/ml). (I) BMSCs were cotransfected with the indicated siRNA and TOP/FOP Flash reporter plasmid. (J) ChIP assays on the promoter regions of the Runx2 and Osterix genes were performed in control and Bcl-3-silenced BMSCs. The data are presented as the mean ± SD. **P < .01; ****P < .0001 vs. control group. Statistical analysis was performed using two-way ANOVA. Primary antibodies: GAPDH, Abcam(ab181602); Lamin B1, Abcam(ab133741); ß-catenin, Proteintech (51067-2-AP); K49 acetyl-ß-catenin, Cell Signalling Technology(9030); BCL-3, Abcam(ab259832)
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