Nb600 594

Cell pellets were extracted in RIPA lysis buffer (P0013C, Beyotime, China) containing protease inhibitor cocktail (539131, Calbiochem, USA). Protein concentrations were determined by a BCA kit (23225, Pierce, USA). Equal amounts of cell extracts (30 μg total protein) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked in 5% non-fat dried milk in TBS-T at room temperature for 2 h and incubated with indicated primary antibodies at 4 °C overnight. The primary antibodies used in the study were: anti-α-SMA (1 µg/mL, A5228, Sigma-Aldrich), anti-collagen I (1 µg/mL, 600-401-103, Rockland), anti-collagen III (1 µg/mL, NB600-594, Novus), anti-Klf2 (2 μg/mL, ab203591, Abcam), anti-GAPDH (1 µg/mL, 5174, Cell signaling technology). The membranes were washed three times with TBS-T for 10 min, and then incubated with IRDye-680LT Goat Anti-Mouse IgG (H+L) (20 ng/mL,925-68020, Li-Cor, USA) or IRDye-800CW Goat Anti-Rabbit IgG (H+L) (20 ng/mL, 925-32211, Li-Cor, USA) at room temperature for 1 h. After washing in TBS-T for another three times, the membranes were detected by the Odyssey infrared imaging system (Li-Cor, USA). Experiments were repeated three times and the target protein level was quantified by Image J and normalized to internal control.

RAW264. 7 or HGF-1 cells seeded in confocal dishes were fixed with 4% paraformaldehyde at 4°C for 15 minutes after washed by PBS 3 times for 20 minutes each time. The cells were then permeabilized with 0. 2% Triton X-100 for 10 minutes and blocked with 2% BSA for 2 hours, and each step was followed by twice PBS washes to remove residual reagents. After the above steps were completed, primary antibodies were added to the cells and incubated at 4°C overnight. The primary antibodies were added as follows: Piezo1 antibody and CD68 were co-incubated to localize Piezo1 on macrophages; CCR7 (1:200;NB100-712; Novus Biologicals, USA) and CD206 (1:200; #24595; Cell Signaling Technology, USA) were co-incubated to identify macrophage polarization; COL-I (1:200;NB600-408; Novus Biologicals, USA) or COL-III (1:200; NB600-594; Novus Biologicals, USA) was incubated to indicate the existence of collagen in HGF-1. Afterwards, the cells were incubated with donkey anti-goat secondary antibody IgG H&L Alexa Fluor 594 (1:200; ab150132; Abcam, UK) and the goat anti-rabbit IgG H&L Alexa Fluor 488 for one hour at room temperature in the dark. After washing away the secondary antibodies with PBS, DAPI was used to stain the nuclei for 10 minutes in the absence of light. Finally, the images were acquired by the confocal laser scanning microscope and quantitatively analyzed by Image J software.