Autophagy inhibition increases simvastatin-induced cell death in HASM and HAF.

(A&B) Side by side comparison of simvastatin-induced apoptosis (sub-G1 population of the cells) and autophagy (LC3 II formation) in HASM and HAF. Mevalonate cascade inhibition induced an early autophagy response and later apoptotic response. At each time point LC3-II level relative to its time matched control are normalized with time zero LC3-II levels to highlight temporal trends. Data represent means ± s.e. mean of three independent experiments, using 3 different cell lines. Cell viability was measured in cultured HASM and HAF after treatment with 10 µM simvastatin for 96 hrs with and without pretreatment with the following inhibitors of autophagy: (C, E) 1.25 or 2.5 mM 3-MA; (D, F) 0.01 or 0.02 µM Baf-A1. Results are expressed as percentage of corresponding time point control and represent the means ± SD of 12 independent experiments in three different sets of donor-matched HASM and HAF **, P<0.01; ***, P<0.001. (G) HASM cells co-treated with 10 µM simvastatin for 48 hrs with and without 0.02 µM Baf-A1, photographed under phase contrast microscopy settings. Arrows indicates partially detached cells with condensed morphology.(H) Baf-A1 enhanced LC3-II level in simvastatin treated cells. HASM were treated with simvastatin (10 µM) in indicated time points in presence and absence of Baf-A1. Baf-A1-simvastatin increased LC3-II level compared to simvastatin treatment. (I) 3-MA decreased LC3-II level in simvastatin treated cells. HASM were treated with simvastatin (10 µM) in indicated time points in presence and absence of 3-MA. 3-MA-simvastatin increased LC3-II level compared to simvastatin treatment.