Figure 2

Knockdown of ATF6α expression inhibits adipogenesis. C3H10T1/2 cells stably expressing control shRNA (black bars), shATF6α-1 (dark grey bars) or shATF6α-2 (light grey bars) were induced to differentiate for 8 days. Total RNA collected at day 0, 1, 2, 3, 5 and 8 and extracted using the RNEasy kit (Qiagen). mRNA expression of (a) ATF6α, (b) ATF6β, (d) C/EBPβ, (e) PPARγ, (f) SREBP1c, (g) GLUT4 and (h) aP2 were determined using real-time PCR. Data are normalised to Cyclophilin A and are expressed as means±s.e.m. (n=3). ^* Indicates significant difference in expression from control shRNA at the same timepoint, P<0.05 by ANOVA with post hoc Tukey's test. Inserts show expanded view of early timepoints. (c) Protein lysates were collected from confluent control and ATF6α knockdown cells and western blotted for ATF6α and calnexin as described in Figure 1. (i) Protein lysates were collected from control and ATF6α knockdown cells induced to differentiate for 0, 2, 3, 5 or 8 days, western blotted and aP2 expression quantified as described in Figure 1, n=4, ±s.e.m., ^* indicates significance of P<0.05 with respect to levels at the same time point in control cells. Representative western blots of aP2 and the loading control calnexin (ab13504, Abcam) are shown from the same lysate samples. (j) Control and ATF6α deficient C3H10T1/2 cells were differentiated in IDM medium for 8 days and lipid accumulation assessed by oil-red O staining, as previously described¹³ (upper panels). Alternatively, cells were induced to undergo osteogenesis by treatment with Dulbecco's modified Eagle's medium supplemented with 10% FBS, 10 mM Glycerol-2-phosphate, 50 μg ml⁻¹ ascorbic acid, 300 μg ml⁻¹ BMP2 and 1 μM dexamethasone (lower panels). Alkaline phosphatase activity, indicating osteogenic conversion, was visualised using BCIP/NBT (B1911, Sigma-Aldrich, St Louis, MO, USA).