Ad sh mst1

Ad-sh-Mst1 or control Ad-sh-green fluorescent protein (GFP) was designed and provided by Hanheng Biotechnology (Shanghai) Co., Ltd. The titer of the adenoviruses used in the present study was ~1.58×10¹⁰ PFU/ml. Prior to adenoviral transfection, CMECs (1×10⁶ /ml, 3rd generation) were seeded into six-well plates until they reached a confluency of 80%. The cells were then incubated with Ad-sh-Mst1 at an MOI of 200 for 48 h at 37°C. Fluorescence microscopy was used to observe GFP expression. The mRNA level of Mst1 was further measured to evaluate the effectiveness of adenoviral transfection.

Cardiac microvascular endothelial cells (CMECs; C-12285, PromoCell) were cultured in endothelial cell growth medium MV (C-22020, PromeCell) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO₂ humidified incubator. Ang II (100 µM, 24 h) was used to induce the in vitro HP cell model as previously described (17 (link),18 (link)). Mst1 silencing was achieved by transfecting Ad-sh-Mst1 [MOI: 200; Hanheng Biotechnology (Shanghai) Co., Ltd.] into the cells. The cultured CMECs were treated with 50 nM bafilomycin A1 (MiilliporeSigma) for 2 h to evaluate the autophagic flux.