Fluorescently tagged secondary antibody

Cells were seeded onto Matrigel-coated PAGs and glass coverslips at a density of 7–9 × 10⁶ in supplemented media. Media was aspirated and adherent cells were washed with 1 × PBS, fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) (v/v) for 10 min at room temperature, then washed three times with 1 × PBS and permeabilised for 5 min in permeabilising solution—0.2% Triton X-100 (v/v) (Sigma-Aldrich) in wash buffer (0.5% bovine serum albumin (BSA, ThermoFisher, (w/v) in PBS).Permeabilising solution was aspirated, and the cells were washed three times with wash buffer. For TAZ staining, samples were first blocked for 1 h at room temperature with 5% BSA in normal serum (10% Donkey serum (sigma-Aldrich) (v/v) in 1 × PBS). Cells were then incubated with a primary antibody targeting CTGF (1:1000, Ab6692, Abcam) or TAZ (1:200; Ab110239; Abcam) for 1 h at room temperature followed by washing three times with wash buffer and incubation with fluorescently-tagged secondary antibody (Abcam) diluted at 1:1000 in normal serum for 1 h at room temperature. Cells were counter-stained with DAPI (Sigma-Aldrich) and fluorescently-tagged phalloidin (ThermoFisher) using standard protocols and washed a final three times with wash buffer and twice with distilled water before mounting onto microscope slides.

To detect DNA damage and the proliferative activity of cell γ-H2AX, Ki67 immunofluorescence staining was performed. Specifically, cells were fixed with 4% paraformaldehyde and exposed to 0.25% Triton X-100 (Sigma, USA) in PBS solution for 10 min at room temperature. The cells were then blocked with 5% BSA solution for 30 min and incubated with the primary antibody anti-γH2AX or anti-Ki67 (1 : 100, Abcam) overnight at 4°C. After washing the cells with PBS, the cells were further incubated with a fluorescently tagged secondary antibody (1 : 500, Abcam) for 1 h at room temperature in the dark. Subsequently, Hoechst 33342 (20 μg/ml, Yeasen, China) was used to stain the nuclei. Finally, immunofluorescence staining was observed under a fluorescence microscope (Leica DMi8, Germany).