All siRNA pools were purchased from Sigma Pro ligo. The siRNA sequences are listed in Additional file 1. Two siRNA pools for KEAP1 and all three compound library pools for NRF2 were comprised of 10 non redundant siRNAs at low concentration which has been shown to result in superior specificity while retaining potent tar get message knockdown compared to less complex pools at higher concentrations. The final pool for KEAP1 was generated through the esiRNA technique. siRNA transfection and RNA preparation for microarray Briefly, endoribonuclease prepared short interfering RNAs or siRNA pools for NRF2 and KEAP1 were incubated with Hiperfect re agent in basal media with no serum or antibiotics and allowed to complex for 10 min at room temperature.
During this incubation, normal human lung fibroblasts were plated in T25 flasks in media con taining 2% serum and growth factors but no antibiotics. The complex was then added to the cell suspension of each well. Cells were then incubated for 30 hr or 48 hr in a humidified incubator. At the end of the incubation period, the culture medium was removed and the cells were lysed by direct resuspension in Trizol reagent. Crude total RNA was isolated from Trizol dissolved samples and purified using the RNAeasy kit as per the manufacturers instructions. RNA concentration was measured using a NanoDrop ND 1000, and RNA in tegrity was determined with a 2100 Bioanalyzer. Samples displaying a RNA integrity number greater than 8 were used for profiling. Affymetrix GeneChip experiment Samples were amplified and labelled using a custom automated version of the RT IVT protocol and reagents provided by Affymetrix.
Hybridization, labelling and scanning were completed following the manufacturers recommendations. For data analysis, we used the mock transfected sample as the reference to compare with all other time matched samples to obtain the ratio data. Merck Affymetrix human custom arrays monitoring 43,737 individual transcripts were used. Raw intensity was normalized using the RMA algorithm. En richment for biological processes was performed by comparing each gene signature against the public gene collections Gene Ontology, KEGG, Swissprot and Pan ther families. Enrichment P values were corrected for multiple testing by using Bonferroni correction. Pathway analysis was performed using Ingenuity Pathway Analysis.
NRF2 and KEAP1 siRNA transfection for Q PCR and chemokine cytokine mesurements Briefly, siRNA pools for NRF2 and KEAP1 were incu bated with Hiperfect reagent in basal media with no serum or antibiotics and allowed to complex for 10min at room temperature. During this incubation, normal human lung fibroblasts were plated in 24 well or 96 well plates, at 4��104 or 2��104 cells GSK-3 well, respectively, with 2% serum but no antibiotics. The complex was then added to the cell sus pension for each well. Cells were then incubated for 48 hrs in a hu midified incubator.