Sc 53142

To assess vascular remodeling, dual staining for monoclonal mouse anti-αSMA (Santa Cruz, # SC53142, clone: B4 1:200, St. Louis, MO, USA) and polyclonal rabbit anti-von Willebrand factor (vWF; Abcam, #ab6994, 1:200, Cambridge, UK) as an indicator for SMC and endothelial cells, respectively, was performed with paraffin-embedded lung, kidney and liver sections (3 µm). Sections were counterstained with hematoxylin and examined by light microscopy using either ten random fields of view per lung section or a computerized morphometric system (Leica QWin V3, Wetzlar, Germany) for assessing medial wall thickness and the degree (none, partial or complete) of muscularization of small peripheral pulmonary arteries (<100 µm). The number of microvessels (<100 µm) was assessed in ten random fields of view per lung section. In total, 3–5 sections per animal were analyzed.

Cellular protein samples were isolated with a Protein Extraction Kit (Qiagen, GmbH, Hilden, Germany) according to the kit's manual and supplemented with a protease inhibitor cocktail (Abcam, Cambridge, UK). Protein samples were separated with 10% SDS-PAGE gel and were transferred to a polyvinylidene fluoride hydrophobic membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skimmed milk (Solarbio, Beijing, China) overnight at 4°C to cover the nonspecific binding sites. Then the membrane was inoculated with the rabbit anti-mouse KLF-4 (BM0485, Abzoom Biolabs, Dallas, TX, USA; 1 : 300), PAI-1 (sc-8979, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), E-cadherin (sc-7870, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 500), Collagen I (ab21286, Abcam, Cambridge, UK; 1 : 200), mouse anti-mouse α-Smooth Muscle Actin (α-SMA) (sc-53142, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), or rabbit anti-mouse β-actin (sc-7210, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 800) at room temperature for 2 hours. The specific binding to each protein marker was presented with the incubation with the peroxidase-conjugated secondary antibody (Promega, Madison, WI, USA) and the electrochemiluminescence (ECL) detection system (Amersham, Uppsala, Sweden). The protein level was presented as a ratio to β-actin.

Immunohistochemistry was performed using the universal two-step immunohistochemical method. The expression of α-SMA in liver tissues was examined was detected using rabbit monoclonal antibody against α-SMA (catalog # sc-53,142; dilutions 1:200; Santa Cruz Biotechnology, Santa Cruz, CA). PBS was added instead of the primary antibody in the negative control.

Tissues that were paraformaldehyde-fixed overnight and then paraffin-embedded and sliced. Sections were stained with hematoxylin and eosin (H&E) or Picrosirius red. For immunohistochemistry staining, sections were incubated with primary antibody against HDAC5 (Abcam, ab55403, 1:200) or α-SMA (Santa Cruz, sc-53142, 1:200) overnight at 4 °C and on the next day, sections were incubated with HRP-conjugated secondary antibody and counterstained with hematoxylin and developed with diaminobenzidine. Image-Pro Plus 6.0 software was used for quantitative analysis.

Tissue sections were subjected to antigen retrieval and blocking as described above, followed by incubation with a rabbit anti-ANGPTL8 antibody (ab180915, Abcam) together with mouse anti-α-SMA (sc-53142, Santa Cruz), anti-MAC-2 (ab278071, Abcam), and anti-CD3 (sc-20047, Santa Cruz) antibodies in PBS containing 0.2% Triton X-100 at 4°C overnight. After three washes with the same buffer, sections were incubated with Alexa Fluor 555-conjugated and Alexa Fluor 488-conjugated antibodies against rat and mouse or rabbit IgG, respectively, for 2 h at room temperature. After washing section, fluorescent images were acquired with a Nikon TS2-S-SM microscope equipped with a Nikon DS-Qi2 camera.

Cells were fixed in 4% paraformaldehyde, then permeabilized with 0.3% Triton X-100 (Byotime, ST795), and blocked with goat serum (Dalian Meilunbio, MB4508). Thereafter, cells were incubated with primary antibodies for S100A4 (1:200), p-STAT3 (Tyr705, 1:500) or α- SMA (Santa Cruz, sc-53142, 1:50) respectively, overnight at 4°C and then combined with TRITC- conjugated goat anti-mouse IgG (ZSGB-BIO, ZF-0313, 1:100) or FITC-conjugated goat anti-rabbit IgG (ZSGB-BIO, ZF-0311; 1:100). Cell nuclei were stained with DAPI (Byotime, C1005, 1:100) for 5 min at room temperature. The fluorescence images were captured by Inverted fluorescence Microscope (Leica, DMI3000B).