Fig. 6. KMS99220 exerts early anti-inflammatory effects independent of Nrf2. (A) BV2 cells were treated with 10 µM KMS99220 for 0.5 or 1 h, and RT-PCR was performed against HO-1, NQO1, GCLM and GCLC using GAPDH as a loading control. (B–D) BV2 cells were transfected with siRNA for control or Nrf2 for 24 h. (B) RT-PCR against Nrf2 was performed to confirm the knockdown. (C) The cells were treated with 10 µM KMS99220 for 1 h, and then with 0.2 µg/ml LPS for 0.5 h. Western blot analysis was performed against HO-1 and p-IκB using β-actin as a loading control. (D) The Nrf2 knockdown cells were exposed to 10 µM KMS99220 and/or 0.2 µg/ml LPS for 6 h, and RT-PCR was performed against iNOS. (E) RT-PCR against Nrf2 was performed to confirm the knockdown in the GFPsh cells and Nrf2sh cells. (F) GFPsh cells and Nrf2sh cells were treated with 10 µM KMS99220 for 1 h, and then with 0.2 µg/ml LPS for 0.5 h. Western blot analysis was performed against HO-1 and p-IκB. (G) GFPsh cells and Nrf2sh cells were exposed to 10 µM KMS99220 and/or 0.2 µg/ml LPS for 6 h, and RT-PCR was performed against iNOS. For quantitation, values obtained from densitometry were normalized against respective GAPDH or β-actin, and expressed as fold of untreated (A), control siRNA (B), GFPsh (E), or respective LPS-treated control (C, D, F and G).