Figure 4.
Figure 4. NFE2L2 (NRF2) and GSH synthetic genes are reduced in expression in TSC-deficient cells in response to THZ1 treatment. (A) The ratio between mRNA levels assessed by RNA-seq for 30 nM THZ1–treated HCV.29.TSC1− cells in comparison to 30 nM THZ1–treated HCV.29.TSC1-intact cells, each treated for 6 h. The majority of transcripts are reduced in expression; red arrow (dot) indicates NFE2L2 (NRF2), which is reduced by 87%. Average of two independent samples assessed by RNA-seq. (B) H3K27ac ChIP-seq data for three kidney angiomyolipomas demonstrates read depth (bigWig data) in or near NFE2L2. H3K27ac ChIP-seq data from a normal kidney are also shown as control for comparison. All angiomyolipomas had loss of TSC2. A superenhancer region identified by ROSE is highlighted in red. (C) Relative mRNA expression of the indicated genes in HCV.29.TSC1− cells treated with vehicle (CTRL) or 30 nM THZ1 for the indicated periods of time. Gene expression is normalized to actin. Each data point represents the mean ± SEM of three independent experiments. (D) Immunoblot analysis of NRF2, KEAP1, and GSH synthetic enzymes in HCV.29.TSC1− cells treated with 30 nM THZ1 for varying periods of time (hours). β-Actin serves as a loading control (two independent experiments performed). (E) Q-RT-PCR analysis of CDK7 expression and NRF2 after CDK7 KO (CDK7.KO) treated with or without THZ1 compared with control (EV) in HCV.29.TSC1− and HCV.29.TSC1+ (top). Immunoblot analysis of lysates is shown at bottom. Each data point represents the mean ± SEM of three independent experiments. Note that data for two different gRNAs is shown, and that THZ1 has no effect on NRF2 levels. (F) NRF2 and pS6s240/244, assessed by IHC, is shown in normal kidney and an angiomyolipoma that has loss of TSC2 (by sequencing). Nuclear localization of NRF2 is indicated by arrows. Representative image taken from one of three patient angiomyolipoma slides; at least eight images per slide were analyzed for each condition (Table S7). Scale bar, 50 µm. (G) Phase-contrast images (left) of HCV.29.TSC1− and HCV.29.TSC1-intact cells transfected with control siRNA (si.CTRL) or siRNA against NRF2 (si.NRF2) after 3 d (top). Two independent experiments were performed; ≥10 images per field were analyzed for each condition. Scale bar, 100 µm. Immunoblot analysis of lysates is shown at bottom (two independent experiments performed). (H) PicoGreen cell number assay at 5 d in HCV.29.TSC1− and HCV.29.TSC1+ cells after NRF2 silencing, along with siRNA controls (top). Cell growth (Trypan blue assays, 72 h) of HCV.29 after NRF2 silencing compared with control (EV) for indicated time points (bottom). Each data point represents the mean ± SEM of three independent experiments. N.S., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

NFE2L2 (NRF2) and GSH synthetic genes are reduced in expression in TSC-deficient cells in response to THZ1 treatment. (A) The ratio between mRNA levels assessed by RNA-seq for 30 nM THZ1–treated HCV.29.TSC1− cells in comparison to 30 nM THZ1–treated HCV.29.TSC1-intact cells, each treated for 6 h. The majority of transcripts are reduced in expression; red arrow (dot) indicates NFE2L2 (NRF2), which is reduced by 87%. Average of two independent samples assessed by RNA-seq. (B) H3K27ac ChIP-seq data for three kidney angiomyolipomas demonstrates read depth (bigWig data) in or near NFE2L2. H3K27ac ChIP-seq data from a normal kidney are also shown as control for comparison. All angiomyolipomas had loss of TSC2. A superenhancer region identified by ROSE is highlighted in red. (C) Relative mRNA expression of the indicated genes in HCV.29.TSC1− cells treated with vehicle (CTRL) or 30 nM THZ1 for the indicated periods of time. Gene expression is normalized to actin. Each data point represents the mean ± SEM of three independent experiments. (D) Immunoblot analysis of NRF2, KEAP1, and GSH synthetic enzymes in HCV.29.TSC1− cells treated with 30 nM THZ1 for varying periods of time (hours). β-Actin serves as a loading control (two independent experiments performed). (E) Q-RT-PCR analysis of CDK7 expression and NRF2 after CDK7 KO (CDK7.KO) treated with or without THZ1 compared with control (EV) in HCV.29.TSC1− and HCV.29.TSC1+ (top). Immunoblot analysis of lysates is shown at bottom. Each data point represents the mean ± SEM of three independent experiments. Note that data for two different gRNAs is shown, and that THZ1 has no effect on NRF2 levels. (F) NRF2 and pS6s240/244, assessed by IHC, is shown in normal kidney and an angiomyolipoma that has loss of TSC2 (by sequencing). Nuclear localization of NRF2 is indicated by arrows. Representative image taken from one of three patient angiomyolipoma slides; at least eight images per slide were analyzed for each condition (Table S7). Scale bar, 50 µm. (G) Phase-contrast images (left) of HCV.29.TSC1− and HCV.29.TSC1-intact cells transfected with control siRNA (si.CTRL) or siRNA against NRF2 (si.NRF2) after 3 d (top). Two independent experiments were performed; ≥10 images per field were analyzed for each condition. Scale bar, 100 µm. Immunoblot analysis of lysates is shown at bottom (two independent experiments performed). (H) PicoGreen cell number assay at 5 d in HCV.29.TSC1− and HCV.29.TSC1+ cells after NRF2 silencing, along with siRNA controls (top). Cell growth (Trypan blue assays, 72 h) of HCV.29 after NRF2 silencing compared with control (EV) for indicated time points (bottom). Each data point represents the mean ± SEM of three independent experiments. N.S., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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