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( A ) Volcano plot showing dysregulated genes in lung adenocarcinoma tumors (TCGA-LUAD cohort) carrying driver mutations in NFE2L2 or KEAP1 [cutoffs: Log2(FC) = ±0.6 and P -adj ≤ 0.05]. ( B ) Gene Set Enrichment Analysis (GSEA) using Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets in the TCGA-LUAD cohort with mutations in NFE2L2 or KEAP1 . ( C ) mRNA expression of selected genes involved in formaldehyde (FA) detoxification in KEAP1-NFE2L2 mutated and nonmutated (WT) LUAD tumors. ( D ) mRNA expression of selected genes in normal lung and whole blood obtained from GTEX. ( E ) Single-cell RNA-seq data from mouse hematopoietic and progenitor cells (HPC) plotted with kleintools ( https://kleintools.hms.harvard.edu/tools/spring.html ) . ( F ) Representative images of Western blots of selected factors from (A) performed in different cell lines. ( G ) Cystine uptake assay performed in selected cell lines (mean ± SEM, n = 3). ( H ) FA tolerance of A549 cells lacking SLC7A11 (sg SLC7A11 ) complemented with a wild-type copy of SLC7A11 (pSLC7A11) grown in the presence of 100 μM BSO (mean ± SEM, n = 3 and two-way ANOVA, **** P < 0.0001). ( I ) FA tolerance for H1299 cells harboring empty vector (pEV) or pSLC7A11 in the presence of 100 μM BSO [mean ± SEM, n = 3 and two-way ANOVA (Tukey test between untreated (UT) and BSO at each point); for H1299 pEV: **** P < 0.0001 and *** P = 0.0002]. ( J ) Viability of A549 cells carrying sg SLC7A11 and sgNTC (nontargeting control) under cystine deprivation [one-way ANOVA (Tukey test)]. ( K ) Glutathione (GSH) levels of the indicated cell lines maintained during 48 hours under cystine deprivation and exposing to FA during the last 24 hours [one-way ANOVA (Tukey test), ** P = 0.0058]. ( L ) FA tolerance of A549 cells lacking ADH5 in the presence of BSO (100 μM) or ERA (5 μM) [mean ± SEM, n = 6 and two-way ANOVA (Tukey test between UT and BSO or ERA at each point for multiple comparison); * P = 0.0300 and **** P < 0.0001].
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( A ) Volcano plot showing dysregulated genes in lung adenocarcinoma tumors (TCGA-LUAD cohort) carrying driver mutations in NFE2L2 or KEAP1 [cutoffs: Log2(FC) = ±0.6 and P -adj ≤ 0.05]. ( B ) Gene Set Enrichment Analysis (GSEA) using Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets in the TCGA-LUAD cohort with mutations in NFE2L2 or KEAP1 . ( C ) mRNA expression of selected genes involved in formaldehyde (FA) detoxification in KEAP1-NFE2L2 mutated and nonmutated (WT) LUAD tumors. ( D ) mRNA expression of selected genes in normal lung and whole blood obtained from GTEX. ( E ) Single-cell RNA-seq data from mouse hematopoietic and progenitor cells (HPC) plotted with kleintools ( https://kleintools.hms.harvard.edu/tools/spring.html ) . ( F ) Representative images of Western blots of selected factors from (A) performed in different cell lines. ( G ) Cystine uptake assay performed in selected cell lines (mean ± SEM, n = 3). ( H ) FA tolerance of A549 cells lacking SLC7A11 (sg SLC7A11 ) complemented with a wild-type copy of SLC7A11 (pSLC7A11) grown in the presence of 100 μM BSO (mean ± SEM, n = 3 and two-way ANOVA, **** P < 0.0001). ( I ) FA tolerance for H1299 cells harboring empty vector (pEV) or pSLC7A11 in the presence of 100 μM BSO [mean ± SEM, n = 3 and two-way ANOVA (Tukey test between untreated (UT) and BSO at each point); for H1299 pEV: **** P < 0.0001 and *** P = 0.0002]. ( J ) Viability of A549 cells carrying sg SLC7A11 and sgNTC (nontargeting control) under cystine deprivation [one-way ANOVA (Tukey test)]. ( K ) Glutathione (GSH) levels of the indicated cell lines maintained during 48 hours under cystine deprivation and exposing to FA during the last 24 hours [one-way ANOVA (Tukey test), ** P = 0.0058]. ( L ) FA tolerance of A549 cells lacking ADH5 in the presence of BSO (100 μM) or ERA (5 μM) [mean ± SEM, n = 6 and two-way ANOVA (Tukey test between UT and BSO or ERA at each point for multiple comparison); * P = 0.0300 and **** P < 0.0001].

Journal: Science Advances

Article Title: NRF2 coordinates cystine import and glutathione use to sustain ferroptosis resistance and formaldehyde tolerance in cancer

doi: 10.1126/sciadv.aea1431

Figure Lengend Snippet: ( A ) Volcano plot showing dysregulated genes in lung adenocarcinoma tumors (TCGA-LUAD cohort) carrying driver mutations in NFE2L2 or KEAP1 [cutoffs: Log2(FC) = ±0.6 and P -adj ≤ 0.05]. ( B ) Gene Set Enrichment Analysis (GSEA) using Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets in the TCGA-LUAD cohort with mutations in NFE2L2 or KEAP1 . ( C ) mRNA expression of selected genes involved in formaldehyde (FA) detoxification in KEAP1-NFE2L2 mutated and nonmutated (WT) LUAD tumors. ( D ) mRNA expression of selected genes in normal lung and whole blood obtained from GTEX. ( E ) Single-cell RNA-seq data from mouse hematopoietic and progenitor cells (HPC) plotted with kleintools ( https://kleintools.hms.harvard.edu/tools/spring.html ) . ( F ) Representative images of Western blots of selected factors from (A) performed in different cell lines. ( G ) Cystine uptake assay performed in selected cell lines (mean ± SEM, n = 3). ( H ) FA tolerance of A549 cells lacking SLC7A11 (sg SLC7A11 ) complemented with a wild-type copy of SLC7A11 (pSLC7A11) grown in the presence of 100 μM BSO (mean ± SEM, n = 3 and two-way ANOVA, **** P < 0.0001). ( I ) FA tolerance for H1299 cells harboring empty vector (pEV) or pSLC7A11 in the presence of 100 μM BSO [mean ± SEM, n = 3 and two-way ANOVA (Tukey test between untreated (UT) and BSO at each point); for H1299 pEV: **** P < 0.0001 and *** P = 0.0002]. ( J ) Viability of A549 cells carrying sg SLC7A11 and sgNTC (nontargeting control) under cystine deprivation [one-way ANOVA (Tukey test)]. ( K ) Glutathione (GSH) levels of the indicated cell lines maintained during 48 hours under cystine deprivation and exposing to FA during the last 24 hours [one-way ANOVA (Tukey test), ** P = 0.0058]. ( L ) FA tolerance of A549 cells lacking ADH5 in the presence of BSO (100 μM) or ERA (5 μM) [mean ± SEM, n = 6 and two-way ANOVA (Tukey test between UT and BSO or ERA at each point for multiple comparison); * P = 0.0300 and **** P < 0.0001].

Article Snippet: ADH5 -deficient cells were generated in A549 cells stably expressing Cas9 (Addgene, catalog no. 78546) infected with a lentiviral vector coding for an sgRNA against the exon 3 of ADH 5 previously published ( ).

Techniques: Analysis, Expressing, Single Cell, RNA Sequencing, Western Blot, Plasmid Preparation, Control, Comparison