Fig. 5.
10,12 CLA increase of [Ca2+]i, ROS levels and expression of inflammatory and ISR genes are dependent on PLC. A: Cultures of newly differentiated human adipocytes were preloaded with 5 μM Fluo-3 AM. Cultures were injected with vehicle (−), 50 μM 10,12 CLA (circle filled), or 25 μM D609 pretreatment + 50 μM 10,12 CLA (circle open). Emitted fluorescence intensities were collected over time using a multidetection microplate reader. Excitation wavelength was 485 nm, and fluorescence was collected at 528 nm. Means (± SEM; n = 4) are representative of two independent experiments. B: Cultures of newly differentiated human adipocytes were preloaded with DCF for 30 min. Cultures were then treated with BSA vehicle (V) or 50 μM 10,12 CLA (10) alone for 12 h, or pretreated for 30 min with 50 μM D609 followed by 12-h treatment with BSA vehicle (D) or 50 μM 10,12 CLA (10+D). Emitted fluorescence intensities were measured using a multidetection microplate reader. Excitation wavelength was 485 nm, and fluorescence was collected at 528 nm. Means (± SEM; n = 3–12) are representative of three independent experiments. C: Cultures were pretreated for 30 min with 50 μM D609 (D) followed by 12-h treatment with BSA vehicle (V) or 50 μM 10,12 CLA (10). RNA was subsequently isolated and mRNA levels of IL-8, COX-2, ATF-3, GADD34, and GAPDH were measured by real-time PCR. Data are normalized to the vehicle controls. Means (± SEM; n = 2) that do not share a common lower case letter differ (P < 0.05). Data are representative of three independent experiments. One-way ANOVA was used to compare data.