Effects of BAPTA and N-acetylcysteine on PRC induction by uncoupler. A, U2OS cells were plated and after 24 h were either untreated, treated with 40 μm CCCP for 16 h (CCCP), or pretreated with 10 μm BAPTA for 1 h following treatment with 40 μm CCCP for 16 h (CCCP + BAPTA). B, U2OS cells were plated and after 24 h were either untreated, treated with 40 μm CCCP for 16 h, or pretreated with 5 mm N-acetylcysteine for 1 h following treatment with 40 μm CCCP for 16 h (CCCP + N-acetylcysteine). Immunoblots in A and B were probed with rabbit anti-PRC or rabbit anti-Sp1. C, RNA was isolated from cells subjected to the treatments described in B, and the expression of the subset of PRC-dependent inflammatory genes was compared with that of the nucleus- and mitochondrion-encoded respiratory genes using quantitative real time PCR. Results obtained with the CCCP-treated (filled bars) or CCCP + N-acetylcysteine-treated (open bars) cells were normalized to those obtained from vehicle-treated controls. Values are the averages ± S.E. for at least three separate determinations. D, U2OS cells were either untreated or treated with 5 μm MG132 or 20 μm N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) for 8 h.