Fig 1: Dose-dependent effects of LPS/IFN-γ on Tgf-β expression are associated with changes in the cleaved Fmod fragments. (A) Representative WB image showing the contents of NICD, Mmp8, total Fmod, cleaved Fmod, Tgf-β2 and Gapdh in macrophages in response to increasing dose of LPS (0, 5, 10, 25, 50 or 100 ng/ml) for 24 h. (B–F) Quantitation of immunoblots for NICD, Mmp8, total Fmod, cleaved Fmod, Tgf-β2 respectively (average of three replicates shown after normalizing the intensity with Gapdh. (WB = Western blot).
Fig 2: FMOD decreases the expression of Tgf-ß2 signaling pathway in BMDMs. Gene expression of the members of Tgf-ß signaling pathway-Tgf-ß1 (A), Tgf-ß2 (B), Tgf-ßRI (C), Tgf-ßRII (D), Smad2 (E) and Smad3 (F) was determined using real-time PCR in WT-BMDMs pre-treated with DMSO or DAPT (10 µM) for 24 h followed by treatment with FMOD (400 ng/ml) or activated-MMP8 (500 ng/ml) for 24 h. Gene expression was determined using qPCR, normalized to Rpl13a and reported as ratio (mean ± SEM, n = 3 for each group) to DMSO-C or DAPT-C. (G,H) WB of the media concentrated from Apoe-/- and Notch1+/-;Apoe-/- BMDMs treated with TGF-ß2 (human recombinant protein; 5 ng/ml), SB431542 (an inhibitor of activin receptor-like kinase; 15 nM), MMP8 inhibitor (10 nM) or FMOD (100 ng/ml) for 24 h and the quantification. The media was concentrated 50 fold and 20 µl volume was loaded for the WB for secreted Tgf-ß2. Coomassie Brilliant Blue G-250 Dye (CBB) was used to demonstrate equal loading of the media. ***P < 0.001, **p < 0.01, *p < 0.05 (one way ANOVA followed by a Tukey’s multiple comparisons test). (WB = Western blot).
Fig 3: Quantitative real-time PCR validates the RNA-Seq data for fibromodulin (Fmod) and Caspase-4 (Casp4). (A,B) Bar graphs represent fold change in gene expression of Fmod and Casp4 in WT, Notch1+/− BMDMs and WT + DAPT (a potent inhibitor of Notch signaling). BMDMs were pre-treated with DMSO or DAPT for 24 h followed by LPS/IFN-γ or IL4/IL13 for 24 h. (C,D) Bar graphs represent the gene expression of Fmod and Casp4 in Apoe−/−, Notch1+/−;Apoe−/− BMDMs treated with LPS/IFN-γ or IL4/IL13 for 24 h. (E,F) Graphs represent fold change in the expression of Fmod and Casp4 in WT, or Notch1+/− BMDMs 48 h post transfection with Notch1 intracellular domain (NICD) plasmid or siRNA-Notch1. Gene expression was determined using qPCR, normalized to Rpl13a and reported as fold change (mean ± SEM, n = 3 for each group) to WT-C or Apoe−/−C. (G–N) Double immunofluorescence (DIF) staining of BMDMs revealing the pattern of expression of Fmod (G,K), Tgf-β2 (H,L) and their merged images (I,M). Nuclei are shown in blue DAPI staining (J, N). (O) Quantification of DIF staining of BMDMs using Lionheart FX Gene5 software. The data is represented as average intensity of ~200 cells from each group. Original magnification 40 × , Scale bars = 50 µm. ***p < 0.001, **p < 0.01, *p < 0.05 (one way ANOVA followed by a Tukey’s multiple comparisons test comparisons test). (EP = empty plasmid).
Fig 4: Overexpression of NICD increases the total Fmod protein contents in BMDMs and LPS prevents the cleavage of Fmod in a dose dependent manner. (A,B) WB shows the expression of total Fmod in Apoe-/- and Notch1+/-;Apoe-/- BMDMs and quantification of three replicates as determined by Image J. (C,D) WB showing the total Fmod protein content in WT BMDMs 48 h post transfection with empty or NICD plasmids and the quantification of the immunoblots. (E,F) qRT-PCR showing the panel of M1 and M2 genes dysregulated with human recombinant FMOD (400 ng/ml for 24 h). (G) Co-immunoprecipitation showing contents of Tgf-ß2, Fmod and NICD proteins pulled down with Fmod antibody from the WT and Notch1+/- peritoneal macrophages.
Supplier Page from Abcam for Recombinant Human Fibromodulin protein