Ab90776

The PFA fixation and immunostaining processes were performed according to our previously reported methods [6 (link)]. After drug stimulation and washout, cells were fixed with 2% PFA for 15 min at 37 °C then washed twice with PBS. After blocking the cells with blocking buffer [1× PBS Tween-20 (28,352; Thermo Fisher Scientific) containing 1.5% (v/v) bovine serum albumin (A9205; Sigma-Aldrich)] for 30 min at room temperature (22–25 °C), the cells were incubated with a polyclonal antibody against rabbit alpha-actinin (AB90776; Abcam, Cambridge, UK), diluted with blocking buffer to a final concentration of 2 μg/mL, at 4 °C for 18 h. To analyze YAP1 nuclear localization, we used a rabbit monoclonal antibody against active YAP1 (AB205270; Abcam), diluted with blocking buffer to a final concentration of 0.2 μg/mL, at 4 °C for 18 h. After discarding the primary antibody solution, cells were washed twice with PBS and incubated with a secondary antibody solution, including goat anti-rabbit IgG (H + L) secondary antibody (A11034, Alexa Fluor 488, 1:400) and Hoechst 33342 (1:800; Thermo Fisher) at 4 °C for 1 h. Finally, cells were washed twice with PBS, and each well was filled with PBS before performing high-content analysis (HCA).

VSD heart tissue sections (8μm) were used for Ki67 and cyclin D2 staining. The initial slide was selected using a random number generator and every seventh slide after was chosen for microscopy by systematic random selection. The slides were stained overnight with mouse monoclonal antibodies against cardiac troponin T (Abcam, ab8295, 1:200) and an Alexa Fluor® 488-conjugated rabbit anti-Ki67 antibodies (Abcam, ab154201; 1:200) or rabbit antibody against cardiac sarcomeric alpha actinin (Abcam, ab90776, 1:200) and mouse antibody against cyclin D2(Abcam, ab3085,1:200). After three washes, the sections were incubated with Alexa Fluor® 555-conjugated anti-mouse secondary antibodies (Abcam, ab150107; 1:200) or Alexa Fluor® 555-conjugated anti-rabbit secondary antibody (CST, 4409, 1:500) and an Alexa Fluor® 488-conjugated anti-mouse secondary antibody (Abcam, ab150073, 1:500) for 30 min. Three researchers who were blinded to sample identities performed the quantification of cellular Ki67 and cyclin D2 events using either manual counting or digital thresholding (image segmentation and the creation of a binary image from a gray scale) methods. Analysis of the converted binary images was performed using ImageJ (National Institutes of Health).