Fig 2: cANGPTL4 enhanced fibroblast proliferation and migration and reduced fibroblast collagen expression. (a) Representative FACs scatter plots of fibroblasts subjected to 24 h of the indicated treatments, then pulsed with 40 µM BrdU for 30 min prior to FAC S analysis. Fibroblasts were harvested and fixed in 70% ice-cold ethanol, their DNA was denatured with 1.5 M HCl, rinsed to neutralize acidity, then stained with FITC-conjugated anti-BrdU antibody and propidium iodide according to manufacturer’s protocol. Cell populations in the G0/G1, S and G2 phases are gated and the percentages are indicated in parentheses (b,c) Representative images and measurements of percentage wound closure by fibroblast migration. Human primary dermal fibroblasts (1 × 104 cells per insert) were seeded into silicone inserts (ibidi, USA) on a 24-well culture plate (Corning Life Sciences, USA) and allowed to attach overnight in a humidified incubator with 5% CO2 at 37 °C before the removal of the inserts. Fibroblasts were then pre-treated with mitomycin-C (1 mg/mL of culture media) for 2 h to induce cell cycle arrest prior to the start of the scratch assay. Culture medium was replaced with fresh media containing the indicated treatments. The plate was then placed into the Cell-IQ machine for real-time imaging. Wound closure rate was assessed using the on-board analysis software that measured the percentage change in surface area occupied by fibroblasts. (d) Relative mRNA expression of COL1 and COL3 in fibroblasts treated for 24 h with vehicle (saline) or 12 µg/mL cANGPTL4 in the presence or absence of 10 ng/mL TGFß. Ribosomal protein L27 was used as housekeeping gene for normalization of target gene expression. (e) Relative protein expression of COL1A2, COL3A1 and phospho-Smad3 in fibroblasts treated for 24 h with vehicle (saline) or 12 µg/mL cANGPTL4 in the presence or absence of 10 ng/mL TGFß. Bars represent mean densitometry measurements for COL1A2 and COL3A1 (normalized against ß-tubulin), and phospho-Smad3 (normalized against total Smad3). Loading controls were obtained from the same sample lysates. Images are representative of 5 fields of view from n = 5 independent experiments; values are means ± SD, n = 5 independent experiments. **P < 0.01 (Mann-Whitney U-test).

Fig 3: Collagen a1(III) is expressed in the glomerular basement membrane in Alport mice. (A) Dual immunofluorescence analysis was performed on kidney cryosections from 7-week-old wild-type and Alport mice using antibodies for podocin (a slit diaphragm protein) and DDR1 (a collagen receptor). Clear co-localization is apparent, placing DDR1 at the foot processes (bar = 15 µm). (B) Super-resolution structured illumination microscopy (SR-SIM) of dual immunofluorescence staining of a capillary loop from a 7-week-old Alport mouse stained with anti-DDR1 antibodies (in red) and anti-collagen a1(III) antibodies (in green). The adjacent localization (arrowheads) indicates basement membrane localization of collagen a1(III) (bar = 5 µm). (C) RNA-seq results from wild-type and Alport glomeruli show a marked (>20-fold) increase in the expression of Col3a1 mRNA relative to wild-type. These results were confirmed using real-time RT-PCR (data not shown) and microarray analysis [8]. (D) ImageJ analysis of the relative fluorescence for immunostains of wild-type and Alport glomeruli (six independent glomeruli each) shows significant increases of fluorescence intensity in Alport mice. (E) Western blotting shows clear increases in the 139 kDa band corresponding to collagen a1(III). (F) Quantification of the relative band intensity for triplicate blots of wild-type and Alport mouse glomeruli indicates significantly elevated abundance of collagen a1(III) in Alport glomeruli relative to wild-type, consistent with the RNA-seq findings. *p < 0.05, ***p < 0.001.

Fig 4: cANGPTL4 regulates COL1A2 and COL3A1 expression in fibroblasts via a bHLH-dependent signaling mechanism. (a) Immunoblot analysis of indicated proteins from fibroblasts treated with cANGPTL4 (12 µg/mL) in the presence or absence of TGFß1 (10 ng/mL). ß-tubulin, as loading control, was from the same samples. (b,d) Chromatin immunoprecipitation (ChIP) of SBE sites in the human COL1A2 gene promoter region and E-box sites in the human COL1A2 and COL3A1 gene promoter regions using phospho-Smad3 antibodies and scleraxis (ScxA) antibodies respectively. The gene sequences spanning the SBE and E-box sites and a random control sequence were analyzed by PCR from immunoprecipitated chromatin from fibroblasts treated with TGFß1 in the presence or absence of cANGPTL4 (cANG). Pre-immune serum was used as a control. PCR was performed using chromatin before immunoprecipitation as input. M, 100 bp DNA ladder. (c) A schematic diagram showing the relative positions of Enhancer boxes (E-Boxes) and Smad binding elements (SBE) on the human COL1A2 and COL3A1 proximal promoters. Sequences of E-Boxes and SBE are shown. ChIP PCR primers designed specifically to bind scleraxis along this proximal promoter region are indicated. (e) Relative mRNA expression of ID3 in fibroblasts treated with 12 µg/mL of cANGPTL4 and lysed for mRNA at the indicated time points post-treatment. (f) Relative mRNA expression of ID3 in control (Fctrl) and ID3-knockdown (F?ID3) fibroblasts treated for 16 h with 10 ng/mL of TGFß1 (control) or TGFß1 plus 12 µg/mL of cANGPTL4. (g–i) Immunodetection of ID3, COL1A2 and COL3A1 in human dermal fibroblasts subjected to the indicated treatments. (j,k) Immunodetection of ScxA and E47 in the immunoprecipitate of ID3. ß-tubulin or IgG, as loading controls, were from the same samples/immunoprecipitates. Cropped images are representative of immunoblots from n = 5 independent experiments; full-length immunoblots scanned using the Li-Cor Odyssey® CLx system can be found in Supplementary Fig. S4a–g; values represent mean ± SD from n = 5 independent experiments. *P < 0.05, **P < 0.01,***P < 0.001 (Mann-Whitney U-test).

Fig 5: KDM5B deficiency prevents pressure overload-induced cardiac dysfunction and cardiac fibrosis.a Representative echocardiographic M-mode images of left ventricles from KDM5B-KO or littermate control WT mice on Day 28 after AngII or normal saline (NS) infusion. b, c Echocardiographic measurement of the LVEF (b) and LVFS (c) of KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). d, e The ratio of heart weight to body weight (HW/BW) (d) and the ratio of heart weight to tibia length (HW/TL) (e) of KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). f–i Representative Masson’s trichrome images and quantitation of perivascular (f, g) or interstitial (h, i) fibrosis in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). Scale bar, 200 µm (left), 100 µm (right). j Q-PCR analysis of Acta2, Col1a1, Col3a1 and Ctgf mRNA expression levels in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). k Immunoblot analysis of Col I and Col III protein expression in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion. l Representative immunofluorescence staining of a-SMA (red) and Col III (green) in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII infusion. Scale bar, 50 µm. *p < 0.05, **p < 0.01. Unpaired Student’s t test (g, i) or one-way ANOVA (b–e, j) was performed.