Fig. 3.

Time course of CLA–mediated increase in stress-related and inflammatory gene expression. A: Cultures of newly differentiated human adipocytes were serum-starved for 24 h and then treated for 6, 12, 24, or 48 h with BSA vehicle (circle open), 30 μM cis-9, trans-11 CLA (square open), or 30 μM trans-10, cis-12 CLA (triangle up filled), and then harvested. RNA was isolated and the mRNA levels of ATF3, CHOP, GADD34, IL-6, IL-8, COX-2, and GAPDH (load control) were measured using real-time PCR. Means (± SEM; n = 2) with asterisks (*) differ significantly (P < 0.05) from the BSA controls at each time point, and are representative of at least two independent experiments. Statistical analyses were performed for data testing the main effects of treatment (BSA, 10,12 CLA, 9,11 CLA) and time (6, 12, 24, 48 h) and their interaction. B: To measure indicators of mitochondrial stress (i.e., less JC-1 staining and more cytoplasmic cytochrome C), cultures of human adipocytes were treated vehicle control (NT), the positive controls CCCP (20 μM), antimycin A (AM, 20 nM), or thapsigargin (Tg), or BSA vehicle (V), 50 μM 9,11 CLA (9), or 50 μM 10,12 CLA (10) for 12 h (JC-1 staining) or 24 h (cytochrome C [cyto C] release). For the JC-1 assay, fluorescence was measured at 590 and 530 nm. Means (± SE; n = 6–12) that do not share a common lowercase letter differ (P < 0.05). Data are representative of two independent experiments. One-way ANOVA was used to compare data. For the cytochrome C release assay, cells were harvested and subcellular fractionation was performed to collect mitochondrial and cytosolic proteins. The cytosolic fraction was immunoblotted for cytochrome C and GAPDH. Data are representative of one independent experiment.