Extended Data Fig. 5: ECM stiffness regulates resistance to oxidative stress.
a, Representative gating scheme for quantification of CytoGrx1-roGFP2 by flow cytometry. Cells without CytoGrx1-roGFP2 expression were used as negative control (outlined white area). Cells treated with cumene hydroperoxide (CUM, purple) for 10 min. to induce glutathione oxidation were used as positive control (n = 4 biologically independent samples pooled across two independent experiments for each bar; unpaired two-tailed Student’s t-test). b, Cell survival by the resazurin assay in MCF10A cells with the indicated knockdowns, plated on stiff or soft Matrigel substrata, and treated for 48 h with Cumene hydroperoxide (CUM). Mean cell number in controls was set to 100%, and all other samples are relative to this (n = 12 biologically independent samples pooled across two independent experiments for each bar; Dunnet’s tests). c, Cell survival of MCF10A-RAS cells transfected with the indicated KEAP1 siRNAs and treated for 48 h with Cumene hydroperoxide (CUM). Mean cell number in Stiff controls was set to 100%, and all other samples are relative to this (n = 12 biologically independent samples pooled across two independent experiments for each bar; Dunnet’s test). d, qPCR to control for efficient knockdown of KEAP1 in MCF10A-RAS. mRNA expression data are relative to GAPDH levels; mean level in the control was set to 1, and other samples are relative to this (n = 4 biologically independent samples from two independent experiments; Dunnet’s test). e, Immunoblotting for endogenous KEAP1 from extracts of MCF10A-RAS cells transfected with the indicated siRNAs. Equal total proteins were loaded in each lane, and GAPDH was used as loading control. Images are representative of two independent experiments with similar results. Unprocessed blots in Source Data Extended Data Fig. 5. f, Cell survival of MCF10A-RAS cells transfected with the indicated NRF1 siRNA, plated on stiff or soft Matrigel substrata, and treated for 48 h with Cumene hydroperoxide (CUM). Mean cell number in Stiff controls was set to 100%, and all other samples are relative to this (n = 12 biologically independent samples pooled across two independent experiments for each bar; unpaired two-tailed Student’s t-tests). g, qPCR for NFE2L1 (encoding for NRF1) and NFE2L2 (encoding for NRF2) expression levels in MCF10A-RAS transfected with NRF1 siRNAs. mRNA expression data are relative to GAPDH levels; mean level in the control was set to 1, and other samples are relative to this (n = 6 biologically independent samples from two independent experiments; Dunnet’s test). h,i, Cell survival of MCF10A-RAS cells treated for 48 h with Erastin (ERA, g) or RSL3 (h) alone and together with the anti-ferroptosis compounds Liproxstatin-1 (LIP) or Ferrostatin (FER). Mean cell number in Stiff controls was set to 100%, and all other samples are relative to this (in h n = 7 (VEHICLE DMSO, ERASTIN 10μM FER, ERASTIN 10μM LIP, ERASTIN 30μM DMSO) n = 6 (ERASTIN 10μM DMSO) n = 8 (all other conditions) biologically independent samples pooled from a single experiment; in i n = 8 (VEHICLE) n = 6 (RSL3 5μM LIP) n = 7 (all other conditions) biologically independent samples pooled from a single experiment; Dunnet’s tests). j, qPCR for YAP/TAZ targets in MCF10A-RAS cells used as a control for differential stiffness of the Matrigel substrata within the extracellular flux analyser plates (see Fig. 4f). mRNA expression data are relative to GAPDH levels; mean level in the control was set to 1, and other samples are relative to this (n = 6 biologically independent samples from three independent experiments; unpaired two-tailed Student’s t-tests). k, qPCR for YAP/TAZ targets in D2.0R cells used as a control for differential stiffness of the Matrigel substrata within the extracellular flux analyser plates (see Fig. 4g). mRNA expression data are relative to GAPDH levels; mean level in the control was set to 1, and other samples are relative to this (n = 4 biologically independent samples from two independent experiments; unpaired two-tailed Student’s t-tests). Data are mean and single points. See Source Data Extended Data Fig. 5.
