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. 2020 Oct 26;40(22):e00377-20. doi: 10.1128/MCB.00377-20

FIG 4.

FIG 4

Nrf2-dependent changes in the cellular phenotype are not required for the synthetic lethal effect. (A) The relative expression of the four HSP90 homologues in WT-GFP and Keap1 KO-mCherry cells as measured by qPCR. (B) The relative expression of the Nrf2 target genes NQO1, GCLM, and GSTM3 in response to 0.1% DMSO or 100 nM 17-AAG treatment for 24 h in WT-GFP and Keap1 KO-mCherry cells as measured by qPCR. (C) The relative expression of the two β-TrCP homologues BTRC and FBWX11 and the NRF2 target genes NQO1, HO1, GSTP1, and GCLM in A549 cells after treatment with an siRNA targeting β-TrCP1/2, or a scrambled control, as measured by qPCR. (D) The relative survival of A549 cells after 4 days of treatment with an siRNA targeting β-TrCP1/2 or a scrambled control. Note that there is no change in cell survival upon hyperactivation of NRF2. (E) The ratio of mCherry to GFP fluorescence from cocultured WT-GFP and Keap1 KO-mCherry cells after 8 days of treatment with either 0.1% DMSO or 100 nM 17-AAG and cotreatment with the indicated concentrations of the antioxidant NAC. Note that 17-AAG kills the vast majority of Keap1 KO cells under all conditions, and therefore, the ratio of mCherry to GFP is low in both the presence and absence of NAC. (F) The ratio of mCherry to GFP fluorescence from cocultured WT-GFP and Keap1 KO-mCherry cells after 8 days of treatment with either 0.1% DMSO or 100 nM 17-AAG, cultured in media containing the indicated percentages of growth serum. (G) Viabilities, determined by fluorescence intensity relative to the DMSO control, of cocultured WT-GFP and Keap1-Nrf2 DKO-mCherry cells exposed to the indicated concentrations of 17-AAG for 8 days. (H) Visualization of the cocultured WT-GFP and Keap1-Nrf2 DKO-mCherry cells shows that in cocultures treated with 800 nM 17-AAG, the mCherry signal from the DKO cells dominates the surface of the microplate well. Scale bars = 300 μm. (I) Viabilities, determined by fluorescence intensity, of cocultured WT-GFP and Keap1 KO-mCherry cells exposed to combinations of 0.1% DMSO, 100 nM 17-AAG, and 2 μM Kribb11 (HSF1 inhibitor). *, P < 0.05. (J) Relative death of A549 and COR-L105 cells exposed to 0.1% DMSO or 200 nM 17-AAG for 3 days, as determined using the CellTox membrane permeability assay.