7KCh-induced inflammation is independent of PI3K-Akt activation.

<p>ARPE19 cells were treated with 8 µM 7KCh for 24 hr and the mRNA inductions of the inflammatory markers were measured by qRT-PCR <b>(a)</b> Measurements (mean ± s.d., <i>n</i>≥9) with and without 10 µM LY294002. LY294002 significantly suppressed the mRNA induction of five of the six inflammatory markers (VEGF: 4.2 to 2.3 fold, IL-1β: 5.6 to 2.7 fold, IL-8: 4.1 to 2.6 fold, CHOP: 15.7 to 9.0 fold, and GRP78: 5.0 to 2.2 fold) but had little effect on the the IL-6 induction (16.0 to 18.1 fold). <b>(b)</b> Measurements (mean ± s.d., <i>n</i> = 5) with and without 1 µM Wortmannin. Wortmannin did not inhibit the induction of the inflammatory markers, instead it increased the mRNA induction of IL-1β (5.0 to 6.9 fold), IL-6 (12.3 to 40.6 fold), and IL-8 (3.5 to 6.8 fold). It had no effect on VEGF, CHOP or GRP78. <b>(c)</b> Immunoblot demonstrating the effect of the knockdown of P110α (catalytic subunit of PI3K) with a siRNA. The P110α knockdown inhibited the phosphorylation of Akt induced by 6 hr of 8 µM 7KCh treatment. The 6 hr time point was determined in prior experiments as the peak in 7KCh-induced Akt phosphorylation. A scrambled siRNA was used as control. <b>(d)</b> Measurements (qRT-PCR) (mean ± s.d., <i>n</i> = 3) of the inflammatory markers with and without P110α knockdown. P110α knockdown did not change the mRNA induction of VEGF, IL-1β, IL-6, and enhanced the induction of IL-8 (3.3 to 4.3 fold), CHOP (15.7 to 20.4 fold), and GRP78 (6.8 to 8.1 fold). *<i>p</i><0.05, two-tailed Student's t-test.