Ccnd1

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used as described^{13 (link),29 (link),30 (link)} to assess MRD in heparinized BM aspirates pooled from four sites (2–2.5 ml/site, from bilateral posterior and anterior iliac crests). The MRD marker panel included cyclin D1 (CCND1), GD2 synthase (B4GALNT1), ISL LIM homeobox 1 (ISL1) and paired-like homeobox 2b (PHOX2B). β2 microglobulin (β2M) was used as the endogenous control, and NB cell line NMB7 as the positive control. Each sample was quantified using the comparative CT method as fold-difference relative to NMB7. All gene expression assays were from Applied Biosystems: CCND1: Hs00277039_m1; B4GALNT1: Hs00155195_m1; ISL1: Hs00158126_m1; PHOX2B: Hs00243679_m1; β2M: 4326319E. For each marker, positivity was defined as greater than the upper limit of normal. All samples were run in duplicates. MRD panel positivity was defined as any one of 4 markers being positive, and negativity as all 4 markers being negative.

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.