Collagen 1α1

After being deparaffinized at 65°C for 2 h, the slides were cooled for 1 h at room temperature. After that, the slides were blocked with 10% goat serum at room temperature for 50 min, and then incubated with primary antibodies: collagen 1α1 (Cell Signaling Technology, 1:200), fibronectin (Abcam, 1:500) and α-SMA (Abcam, 1:200) overnight at 4°C. Later on, the slides were incubated with corresponding secondary antibodies for 20 min. A fluorescence microscope was used to observed histological images. Image-ProPlus software used to quantitatively analyze the expression of each protein.

Total proteins were quantified using the BCA assay kit (Beyotime), and equal amounts of protein (30 μg) were separated by 10% SDS-PAGE. After that, proteins were transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk in tris-buffered saline with tween-20 (TBST) for 1 h, the membranes were incubated with primary antibodies: collagen 1α1 (1:1000, Cell Signaling Technology), fibronectin (1:1000, Abcam), alpha-smooth muscle actin (α-SMA; 1:1000, Abcam), TSC1 (1:1000, Abcam), phosphorylated-mTOR complex 1 (p-mTORC1; 1:1000, Cell Signaling Technology), mTORC1 (1:1000, Cell Signaling Technology), phosphorylated-p70 ribosomal s6 kinase (p-p70S6K; 1:1000, Cell Signaling Technology), p70S6K (1:1000, Cell Signaling Technology), phosphorylated-eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1; 1:1000, Cell Signaling Technology), 4E-BP1 (1:1000, Cell Signaling Technology), β-actin (1:1000, Abcam) at 4°C overnight. Later on, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h, and then the protein bands were visualized using the enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific).