Fig. 2.

PKC knockdown prevents the increase in NOX activity and ROS generation in HCAECs exposed to Ca²⁺ionophore ionomycin. Cells were incubated with Fluo4-AM for 30 min, followed by 30 min de-esterification, Ca²⁺ was measured using live cell imaging. Briefly, cells were imaged every 5 s for five times to obtain a baseline value of total fluorescence intensity (TFI0). Afterwards, 0.2 μM ionomycin or 0.02 % DMSO (Veh) was loaded on the cells and the changes of fluorescence intensities of cells were recorded every 5 s for 5 min. The increase in intracellular Ca²⁺ was quantified using the area under curve (AUC) (A, n = 5). Representative images are shown in Figs. S16–A. To explore the role of PKC-β in Ca²⁺ related oxidative stress, PKC-β knock down was performed using siRNA. Wilde-type HCAECs or PKC-β knockdown cells were subjected to 5 % stretch for 24 h with or without 0.2 μM ionomycin, the study flowchart is shown in Fig. 3B. PKC activity was measured from wild-type cells receiving vehicle or ionomycin (D, n = 4). The efficiency of PKC-β knockdown was validated with qPCR (D, n = 3). NOX activity was measured from cell lysates (E, n = 4) and ROS were quantified using mean fluorescence intensity (MFI) (F, n = 4). Data are presented as mean ± SD. P < 0.05, referred as *; ** for P < 0.01, *** for P < 0.001 and **** for P < 0.0001, were considered statistically significant. ROS data were normalized to 5+Veh group and non-normalized ROS values were presented in Fig. S10 and Table S2.