A16891

Protein was extracted and quantified using RIPA buffer (P0013B, Beyotime, China). Then the protein mixture was centrifuged at 12,000 × rpm for 20 min at 4 °C. BCA protein assay kit (Beyotime, China) was used to detect protein concentration. Protein (50 μg) was subjected to 12% SDS-PAGE and transferred to PVDF membrane (Millipore, USA). Membranes were incubated with the primary antibodies for 18 h at 4 °C. Primary antibodies included COL1A1 (1:1000, A16891, ABclonal, USA), SM22 (1:1000, A6760, ABclonal, USA), AGO2 (1:1000, C34C6, CST, USA), mTOR (1:1000, 7C10, CST, USA), ACE2 (1:1500, ab108252, Abcam, USA), Mdm2 (1:1000, ab259265, Abcam, USA), Ube2n (1:1000, ab25885, Abcam, USA), Uba1 (1:2000, ab264179, Abcam, USA), and GAPDH (1:4000, ab8245, Abcam, USA). HRP-conjugated secondary antibodies were incubated with the membranes for 2 h at room temperature. ECL was used to detect the immunoreactive bands, and densitometry of the blots was analyzed by ImageJ software.

Protein expression of COL1A1 in each group was detected with confocal microscopy (LEICA TCS SP8) after coculture for 1 and 7 days. The samples were fixed with 4% paraformaldehyde, sealed with 10% goat serum (B900780, Proteintech, Wuhan, China), and then incubated overnight with primary antibody (COL1A1, A16891, ABclonal, Wuhan, China) 1:100. After rewarming at room temperature, the samples were incubated with secondary anti-rabbit IgG (AS039, ABclonal, Wuhan, China) 1:200 for 1 h. Subsequently, nuclei were stained using DAPI. Each sample randomly selected 3 vision, and each group of 3 samples, a total of 2 time nodes, measured 18 vision.

Total proteins were separated on 7.5% SDS-PAGE at 60-100 V for 2 h, and then transferred to PVDF membrane (Millipore, USA) at 300 mA for 1.5 h. The membranes were blocked with 5% non-fat dry milk for 1.5 h at room temperature. Next, membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 2 h. The primary antibodies included: rabbit anti-α-SMA (1:1000, ab32575, Abcam), rabbit anti-Col1a1 (1:1000, A16891, ABclonal), rabbit anti-Col3α1 (1:1000, A3795, ABclonal), rabbit anti-β-tubulin (1:1000, #2128, CST), rabbit anti-CD63 (1:1000, ab217345, Abcam), rabbit anti-CD81 (1:1000, ab109201, Abcam), anti-TSG101 (1:1000, ab125011, Abcam), rabbit anti-β-catenin (1:1000, #8480, CST), and rabbit anti-APC (1:1000, ab40778, Abcam). All blots were visualized using the ECL reagent Kit (Millipore Corp, USA). The blot intensities were quantified with Image J software.

MSC(AT)s were seeded in 24-well plates and incubated in a complete medium. After MSC(AT)s reached ~50% confluency, they were prepared for IF staining. HGFs were seeded in 24-well plates and divided into four groups (see Section 4.6, Cell migration assay). For cell IF staining, cells were washed with PBS three times and fixed with 4% paraformaldehyde for 15 min, after which they were treated with 0.1% Triton X-100 for 20 min, blocked by 5% normal goat serum for 30 min at RT, and incubated at 4 °C overnight with antibodies, COL1A1 (A16891, Abclonal, Wuhan, China), IL-1RA (ab124962, Abcam, Cambridge, UK), and FN (A16678, Abclonal, China). The next day, cells were rewarmed at room temperature (RT) for 30 min and incubated with secondary antibodies (ab96899, Abcam, Cambridge, UK or ZLI-0316, ZSGB-BIO, China) for 1 h. After being washed with PBS three times for 5 min each, they were stained with a fluorescent mounting medium with DAPI. Images were acquired with an Olympus microscope (Olympus Corporation, Tokyo, Japan).