Serpina1

The procedure was described previously (24 (link)); the labeled primers, including MYC (Hs00153708), CCND1 (Hs0076553), TFF1 (Hs00907239), CTSD (Hs00157205), PGR (Hs01556702), SERPINA1 (Hs00165475) and CITED1 (Hs00918445) were obtained from Applied Biosystems (Foster City, CA).

MCF-7 cells were seeded in 60 mm dishes at a density of 300,000 cells per dish. Cells were treated with 0.1% DMSO, 100 pM E2, 10 μM LH1092 or 100 nM CDDO-IM (a known Nrf2 activator as a positive control) with or without 100 pM E2 for 16 hours before RNA extraction. To assess the activity of all four Keap1-Nrf2 PPI inhibitors, cells were treated with 0.1% DMSO and 100 pM E2 in the presence or absence of 10 μM test compounds (LH1092, LH1093, LH1095 and LH1101) or 100 nM CDDO-IM for 48 hours before RNA extraction. Total RNA was isolated using the TRIzol reagent from Sigma Aldrich (Saint Louis, MO). The cDNA was synthesized from 1 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Pleasanton, CA). The mRNA levels of genes of interest were measured by RT-qPCR using TaqMan 2X Universal PCR Master Mix (Applied Biosystems, Pleasanton, CA). The primers, PGR (Hs01556702), CCND1 (Hs00277039), CITED1 (Hs00918445), CTSD (Hs00157205), SERPINA1 (Hs00165475), TFF1 (Hs00907239), HO-1 (Hs01110250_m1), NQO1 (Hs00168547), GCLM (Hs00157694), GPX1 (Hs00829989), GPX2 (Hs01591589), and GAPDH (Hs02758991), were obtained from Applied Biosystems (Pleasanton, CA). The ΔΔCt values were calculated from Ct values compared to a reference gene GAPDH. Results are represented as fold changes compared to the DMSO control group.