Fig. 2.

Thiol antioxidant NAC blocks PDTC-induced suppression of ACE2 in H322M cells. (A) H322M cells were treated without (black line) or with (red line) 5 mM NAC or 200 μM PDTC alone or in combination for 24 h. Then, cells were stained using the Cellular ROS assay kit (deep red). Intracellular ROS levels were analyzed with a Becton-Dickinson FACSVerse Flow Cytometer. Cells treated with H₂O₂ for 30 min as a positive control. (B) H322M cells, with or without NAC pretreatment for 30 min, were treated with PDTC for 24 h. Total RNA was collected for ACE2 measurement by real-time qRT-PCR. The data were normalized to GAPDH. Error bars represent the mean ± SD (n = 3). (*P < 0.05, ** P < 0.005, ***P < 0.0005, and n.s. indicates non-significance.) (C) The treated cells were lysed for Western blot analysis of ACE2 and PARP; β-actin was used as a loading control. The bands indicating full-length PARP (f-PARP) and the cleaved fragment (c-PARP) are indicated. (D) H322M cells were electroporated with control or p50 siRNA for 24 h, and then, cells were treated with H₂O₂ for an additional 24 h. The treated cells were lysed for Western blot analysis of ACE2, p50 and PARP; β-actin was used as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)