Ab4069

Cells were collected and lysed using RIPA buffer solution (Abcam Inc., ab156034). Protein content was measured using the BCA method and 30 μg protein aliquots of each sample was separated by SDS-polyacrylamide gel electrophoresis (Solarbio Company, China). Separated proteins were transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA), which were blocked for 1 h and then incubated with primary antibodies specific for Ezrin (1:500) (Abcam Inc., ab4069), YAP₁ (1:500) (Abcam Inc., ab52771), CTGF (1:700) (Proteintech, USA, 23936-1-AP), and GAPDH (1:500) (Abcam Inc., ab181602) overnight at 4°C. Subsequently, secondary antibodies were added and incubated at room temperature for 1 h. The chemiluminescence signals were generated by X-ray film. Finally, protein expression was quantified using Image J software.

Tissues and cells were lysed in RIPA buffer (cat. no. R0020; Solarbio Science and Technology, Beijing, China) to recover total protein. Then the protein quantification was measured using a bicinchoninic acid assay kit (cat. no. 23227; Thermo Fisher Scientific). Twenty micrograms of protein was loaded onto SDS‐PAGE and transferred to polyvinylidene difluoride membranes (cat. no. 1620177; Bio‐Rad). After blocking, protein levels were detected by specific primary antibodies from Abcam (Cambridge, MA, USA) against PDPN (ab236529, 1:1000), ezrin (ab4069, 1:500), Ki67 (ab16667, 1:1000), E‐cadherin (ab40772, 1:10000), N‐cadherin (ab76011, 1:1000), cleaved‐caspase 3 (ab2302, 1:5000), α‐smooth muscle actin (α‐SMA; ab5831, 1:1000), fibroblast activation protein (FAP; ab207178, 1:1000), fibroblast‐specific protein 1 (FSP‐1; ab124805, 1:1000) and GAPDH (ab8245, 1:2500), followed by the corresponding horseradish peroxidase‐conjugated secondary antibodies (ab6721/ab205719, 1:5000) and visualization. GAPDH was used as the internal control.

Forty-eight hours after seeding the cells (HSC-3 and HSC-4: 1 × 10⁵ cells per mL) into a 12-well plate, the culture medium was replaced with a medium containing FR180204 (50 μmol•L^-1). Twenty-four hours after replacing the culture medium, the cells were fixed for 30 min in PBS containing 10% formalin. After membrane permeation treatment for 1 h with PBS containing 0.1% Tween 20, blocking with Histofine Blocking Reagent II (Nichirei Biosciences Inc., Tokyo, Japan) and 10% normal goat serum was carried out for 30 min. The cells were incubated at 4 °C overnight before reacting with the primary antibodies. The primary antibodies used were anti-cortactin rabbit antibody (1:1 000, ab81208; Abcam) and anti-ezrin mouse antibody (1:100, ab4069; Abcam). Cells were then incubated with the secondary antibodies for 1 h at room temperature. The secondary antibody used was Alexa Fluor 488 or 594-labelled anti-mouse IgG antibody or rabbit IgG antibody (Invitrogen). Rhodamine–phalloidin (Invitrogen) was employed for labelling actin. DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (Wako Pure Chemical Industries, Osaka, Japan) was employed for nuclear labelling, and ProLong® Diamond Antifade Mountant (Life Technologies, Carlsbad, CA, USA) was used for mounting. A confocal laser scanning microscope (FV-1000D IX-81; Olympus, Tokyo, Japan) was used to visualize the cells.