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7 protocols using ccnd1

1

RNA Isolation and RT-PCR Protocol

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RNA was isolated from frozen tissues and cell lines using TRIzol Reagent (Invitrogen) and chloroform, and real-time quantitative PCR (RT-PCR) was performed as previously described (21 (link)). CD73 primers and probe sequences, PCR efficiency, and limit of quantification have been previously reported (21 (link)). Taqman primers and probes purchased from Thermo Fisher Scientific include CCND1 Hs00765553; CDH1, Hs01023864; SNAI1, Hs00195591; SNAI2, Hs00950344; ZEB1, Hs00232783; ZEB2, Hs00207691; VIM, Hs009958116. Adenosine receptor primers and probe sets were previously reported and used as described (21 (link)).
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2

Multiplex Immunofluorescence Staining of FFPE Sections

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Formalin-fixed paraffin-embedded (FFPE) 4 µm sections were cut and placed on charged slides. Slides were dried at 65 °C for 2 h. After drying, the slides were placed on the BOND RXm Research Stainer (Leica Biosystems) and deparaffinized with BOND Dewax solution (AR9222, Lecia Biosystems). The multispectral immunofluorescent (mIF) staining process involved serial repetitions of the following for each biomarker: epitope retrieval/stripping with ER1 (citrate buffer pH 6, AR996, Leica Biosystems) or ER2 (Tris-EDTA buffer pH9, AR9640, Leica Biosystems), blocking buffer (AKOYA Biosciences), primary antibody, Opal Polymer HRP secondary antibody (AKOYA Biosciences), Opal Fluorophore (AKOYA Biosciences). All AKOYA reagents used for mIF staining come as a kit (NEL821001KT). Spectral DAPI (AKOYA Biosciences) was applied once slides were removed from the BOND. They were cover slipped using an aqueous method and Diamond antifade mounting medium (Invitrogen ThermoFisher). The mIF panel consisted of the following antibodies: Ki67 (Abcam, ab16667), AE1AE3 (Dako, M3515), CCNE (Abcam, ab33911), CCND1 (ThermoFisher, MA1-39546), CCNA (Abcam, ab32386), RB (Cell Signaling, 9309s), pRB (Cell Signaling, 8516), and MCM2 (BioSb, BSB6334).
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3

Transcriptional Profiling of Stem Cell Markers

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Cells were plated in 25 cm2-angled flasks at 1 × 10^6 cells of confluence, 48 h after treatments, cells were scraped from flasks to extract RNA; the extraction was performed with RNAeasy kit (QUIAGEN, Hilden Germany) according to manufacturer’s instructions. Next cDNA was obtained by retro-transcription assay using Revert Aid Synthesis Kit (Thermofisher, United States). For RT-PCR assays, taqman probes used for: (Hs00610060_m1) SFRP1; (Hs00277039_m1) CCND1; (Hs03986777_s1) FZD4; (Hs02387400_g1) NANOG; (Hs99999903_m1) ACTB; (Hs00153408_m1) MYC; (Hs04260367_gH) POU5F1; (Hs00610344_m1) AXIN2; (Hs00602736_s1) SOX2; (Hs99999901_s1) 18S, and (Hs01547250_m1) LEF1 genes were purchased from Thermofisher (Massachusetts, United States).
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4

Immunohistochemical Analysis of KIT, CDK4, and CCND1

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Immunohistochemical analysis was performed using the Dako EnVision System (Agilent, Santa Clara, CA). Paraffin sections were dewaxed and incubated in 3% H2O2. Antigen retrieval was performed by autoclaving, and the sections were incubated with antibodies against KIT (Agilent), CDK4 (Cell Signaling Technology, Danvers, MA), or CCND1 (Thermo Fisher Scientific, Waltham, MA).
Immunostaining was graded according to an immunoreactivity score (IRS) calculated by multiplying a score based on the percentages of positive cells (0, negative; 1, 1-20%; 2, 21-50%; and 3, 51-100%) by a score based on staining intensity (1, weak; 2, moderate; and 3, strong). The final expression was evaluated as follows: negative (-), IRS 0; low (+), IRS 1 or 2; intermediate (++), IRS 3 or 4; and high (+++), IRS 6 or 9 [17] .
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5

Quantifying Minimal Residual Disease in Neuroblastoma

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Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used as described13 (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.
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6

Primer Analysis of Gene Expression

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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).
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7

Nrf2 Activation and Gene Expression

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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.
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