Fig. 4

AR protein levels are elevated under AD by TRX1 suppression and promote PX-12-induced ROS and loss of viability. a Western blotting for AR. Blots were run using 10 μg of total protein lysate from shRNA-transduced (left) and DMSO or 1 µM PX-12-treated (right) LNAI cells under the indicated conditions. b Cell lines were treated for 48 h with the indicated DMSO or PX-12 doses, under FBS or CSS conditions, prior to assessing viability. Data are representative of n = 2 experiments, each sample run in triplicate per independent experiment. Error bars represent ± SD. c Crystal violet staining of LNAI shAR cells for visual assessment of improved survival under 48 h of PX-12 treatment. d Representative flow cytometric profiles of ROS levels from LNAI shAR cells stained with CM-H2DCF-DA. ROS levels were assessed following an ~6-h treatment with DMSO or the indicated doses of PX-12 under FBS or CSS culture. A rightward shift indicates elevated staining. e Quantitation of ROS fold-changes from (d). At each dose, FL-1H values for CSS were normalized to the counts under FBS conditions. Values were taken from n = 2 independent experiments, each sample run in duplicate. Error bars represent ± SEM. The p-values were determined via a two-tailed Student’s t-test. f Western blot of LNAI cells, mock-treated or treated with either 50 µM H₂O₂ or 250 µM paraquat (PQ) for 24 h. Approximately 20 µg total protein was immunoblotted and probed with the indicated antibodies. g Western blot from total LNAI protein lysates (15 µg) under the indicated time points using doxycycline to induce shRNA expression and probed for AR or Sp1. Note that this blot was run using the same lysates as in Supplementary Fig. 5e. h The indicated samples were analyzed by qPCR and results are represented as fold-change relative to baseline FBS or CSS shGFP values. Fold-changes were calculated from two separate experimental runs, each sample run in triplicate. Error bars represent ± SD. i Western blot of total protein lysates (20 μg) from FBS-cultured LNCaP SB0 and LNAI cells transduced with either the empty pBL vector or TRX1-expressing construct, probed with the indicated antibodies