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. Author manuscript; available in PMC: 2020 Oct 14.
Published in final edited form as: Sci Signal. 2020 Apr 14;13(627):eaba4200. doi: 10.1126/scisignal.aba4200

Fig. 3. NRF2–p53 co-stabilization is enhanced and shNRF2-induced p53 adaptations are preserved in basal-like premalignancy but have different morphometric consequences.

Fig. 3.

(A) Quantitative immunofluorescence of NRF2 and p53 abundance in ECM-attached MCF10DCIS.com cells grown as 3D spheroids. Median-scaled two-color average fluorescence intensities are quantified along with the log-scaled and background-subtracted mutual information (MI) with 90% CI for n = 1832 cells segmented from 70–110 spheroids from two separate 3D cultures. (B) Common changes in transcript abundance identified by RNA sequencing of MCF10A-5E (5E) and MCF10DCIS.com (DCIS.com) cells grown as 3D spheroids with or without NRF2 knockdown. The negative control for shNRF2 was an inducible shGFP (5E) or shLacZ (DCIS.com). Data are log2-transformed Z-scores for genes detected at >5 transcripts per million from n = 4 biological replicates. Enriched gene sets for the BRCA1, ATM, and CHEK2 networks are indicated, with black denoting multiple enrichments. The complete list of enrichments is available in data file S3. (C) Quantification of rounded spheroids (circularity > 0.9) in 3D-cultured MCF10DCIS.com cells with or without NRF2 knockdown. The negative control for shNRF2 was an inducible shLacZ. (D) Quantification of large spheroids (size > e8.5 ~ 5000 μm2) in 3D-cultured MCF10DCIS.com cells with or without p53 disruption. The negative control for p53 constructs was an inducible FLAG-tagged LacZ. (E) Quantification of size and circularity in 3D-cultured MCF10DCIS.com cells with or without NRF2 knockdown, p53 disruption, or both. For (C) to (E), cells with or without inducible perturbations were treated with 1 μg/ml doxycycline for 48 hours, grown as 3D spheroids for 10 days, imaged by brightfield microscopy, and segmented. For (C) and (D), data are mean ± 90% bootstrap-estimated CI from n = 8 biological replicates, with p values by rank-sum test estimated by bootstrapping. For (E), data are mean ± s.e.m. of n = 8 biological replicates. Statistical interaction between NRF2 and p53 perturbations (pint) was assessed by two-way ANOVA with replication. Scale bars are 100 μm.