P38 mapk sirna

Isoniazid and SB203580 (SB) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Mdivi-1, an inhibitor of DRP1, was purchased from Selleck Chemicals (Houston, TX, United States). Tetramethyl rhodamine methyl ester (TMRM), MitoTracker Deep Red FM, Hoechst 33342 and Lipofectamine RNAiMAX were obtained from Invitrogen (Grand Island, NY, United States). p38 MAPK-siRNA, a silencer negative control siRNA, and antibodies against p38 MAPK, phospho-p38 MAPK, NRF1, COX IV, cytochrome c, caspase 9, caspase 3, MFN2, DRP1, acetylated lysine, p-MAPKAPK-2, MAPKAPK-2 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against SIRT1 and Bax were obtained from Abcam (Abcam, Cambridge, United Kingdom). PGC1α antibody and protein A/G-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). All other chemicals were of analytical grade.

MAPK expression was silenced by small RNA interference. The p38 MAPK siRNA (#6564), JNK siRNA (#6232), and scrambled duplex control siRNA (#6568) were purchased from Cell Signaling Technology (Danvers, MA, USA) and used to transfect the cells. The siRNAs were introduced to cultured RAW264.7 cells by using the lipid-mediated transfection reagent RNAiMax (Invitrogen, Carlsbad, CA, USA) in 6-well culture dishes in accordance with the manufacturer’s instructions. The final concentration of siRNAs was 100 nmol/L.

Most of the small interfering RNAs and controls were purchased from Santa Cruz Biotechnology. They are TNF-α siRNA (sc-37216), ATM siRNA (sc-29761), NF-κB p65 siRNA (sc-29410), hMMP3 siRNA (sc-29399), hMMP9 siRNA (sc-29400), hMMP13 siRNA (sc-41559) and control siRNA (sc-37007). p38 MAPK siRNA (#6564) and p44/42 MAPK (Erk1/2) siRNA (#6560) were obtained from Cell Signaling Technology. For siRNA transfection, 30-50% confluent cells were transfected with siRNA using Lipofectamine 2000. The cells were harvested 48 h after transfection. The final concentration for siRNA is 100 nM. The silence effects of indicative siRNA in NCI-H446 cells were validated in Supplementary Figure S8.