Figure 6. JAK2 inhibitor decreases TNFα-mediated inflammation, ROS levels, and interferon signature. Cells were exposed to TNFα alone, TNFα in combination with AG490 (30μM), or AG490 alone for 3 days. (A) Immunoblot detection of p-Ser727-STAT1, p-Tyr705-STAT3, and total STAT1 and STAT3. (B) Secretion of IL6 was estimated by ELISA in culture supernatants from cells treated with TNFα alone, or in combination with AG490, or AG490 alone for 3 days. (C) FACS analysis of ROS levels in cells stimulated either with TNFα, TNFα along with AG490, or AG490 alone for 3 days using DCFDA staining 2',7’-dichlorofluorescein (DCF) positive cells were analyzed. Inhibition of STAT signals modulates senescence. (D) Cell cycle analysis using BrdU and 7- aminoactinomycin D (7-AAD) staining in cells exposed to TNFα, TNFα in combination with AG490, or AG490 alone for 3 days. (E) Percentage of SA-β-gal-positive cells. Quantification of SA-β-gal activity in cells stimulated with TNFα 20ng/ml, TNFα in combination with AG490, or AG490 alone for 3 days. The data represent means of 3 independent counts of 200 cells from 2 independent experiments. (F) Effect of AG490 on cell cycle regulatory proteins. Western analysis performed using cells treated with AG490 for 3 days and blotted against anti-p21, CDK2, and NDC80. Actin serves as loading control.