Col1a

The expression of osteoblast-related proteins was assessed using the ICW assay according to the manufacturer’s instructions. Briefly, cells were fixed in 10% neutral buffered formalin (NBF, Cellpath, Newtown, UK) for 20 minutes prior to be permeabilised with 0.1% Triton X-100 in PBS for 30 min and then blocked with LICOR Odyssey blocking buffer (Li-Cor Biosciences, Cambridge, UK) for 1.5 h at room temperature. The samples were incubated overnight at 4 °C with primary antibodies to ALP (1:300), COL1A (1:200), OCN (1:400) (Abcam, Cambridge, UK) and BMP2 (1:500) (Invitrogen, Life Technologies, Paisley, UK) in Odyssey buffer. Samples were washed in PBS containing 0.1% Tween 20 for five times and then incubated with the IRDye 800CW secondary antibody (1:800) and the CellTag 700 stain (1:500; Li-Cor Biosciences, Cambridge, UK) in the Odyssey blocking buffer for 1 h at room temperature [58 (link)]. The plates were read under the Odyssey SA Imaging System (Li-Cor Biosciences, Cambridge, UK) at both 700 and 800 nm. Relative fluorescent intensity (IRDye 800CW) was normalised with cell number (CellTag 700 stain). The data were quantified using the Image Studio analysis software (Li-Cor Biosciences: v5) [59 (link)].

For immunofluorescence staining of MKL1, FSP1 and Col1a, the tissue slices were incubated with anti-MKL1 (Abcam, 1:200), anti-FSP1 antibodies (Proteintech, 1:100) and anti- Col1a (Abcam, 1:200) respectively, in one humidified chamber at 4°C overnight, followed by incubation with FITC-labeled secondary antibodies at 37°C for 1 h. Then, cell nuclei were stained with DAPI (Sigma) for 15 min. The immune stained images were captured using a confocal microscope (Olympus, Tokyo, Japan).
Immunohistochemistry was performed. Lung paraffin-embedded sections were permeabilized and blocked with 5% BSA, and then incubated with anti-CD68 (Proteintech, 1:100), and anti-CD45 (Proteintech, 1:100) respectively at 4°C overnight, then incubated with secondary antibody (Beyotime Institute of Biotechnology, 1:200) at room temperature for 1 h. Positive immunostaining was visualized by using the diaminobenzidine substrate (DAB, Thermo Fisher, United States) for 1 min. Next hematoxylin was utilized to stain nuclei for 20 s. The immune stained images were captured using a confocal microscope (Olympus, Tokyo, Japan).

The calvarial were decalcified, embedded in paraffin and sectioned (5 μm). Afterward, for immunohistochemical assessment of the osteogenesis, the marker staining of COL1A was observed. Sections were incubated 12 h at 4°C with COL1A (1:500; Abcam). Afterward, the sections were incubated with the biotinylated secondary antibodies (Zhongshan Biotechnology Corporation, Ltd., China).

Mouse fibroblast line NIH-3T3 cells were obtained from ATCC (Manassas, VA) and maintained in complete Dulbecco’s Modified Eagle Medium(DMEM) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units of penicillin and 100 µg of streptomycin at 37 °C in 5% CO₂. Ang II was purchased from Sigma-Aldrich (St. Louis, MO). PI3Kγ inhibitor AS605240 was purchased from Selleckchem (Huston, TX). Akt and p-Akt antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX); while Col1a, fibronectin, α-Sma and Gapdh antibodies were purchased from Abcam (Cambridge, MA). Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo-Fisher Scientific (Pittsburgh, PA).