Journal: Cell reports

Article Title: CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

doi: 10.1016/j.celrep.2024.114345

Figure Lengend Snippet: Figure 4. Ferroptosis resistance and NRF2 activation are conserved functions between BRM and BRG1 and require their ATPase activity (A) Colony-forming assays of KP4 cells overexpressing WT BRG1, catalytic mutants K785R or T910M BRG, or LACZ. Scale bar, 1 mm. Representative of n = 3 biological replicates is depicted. (B) BODIPY-C11 imaging of lipid peroxidation levels in the indicated cells. Each dot represents a cell, and the red lines represent the median. p values were calculated by a Kruskal-Wallis ANOVA with a Dunn’s posttest. Experiments in (A) and (B) were repeated at least 3 times with similar results. (C) Pile-up plots of ATAC-seq-accessible peaks in KP4 cells expressing WT BRG1 versus KR BRG1 mutant, TM BRG1 mutant, or LACZ (n = 2 biological rep- licates). See also Figure S4E and Table S6.

Article Snippet: The following antibodies were used in this study: BRM (Cell Signaling, 11966), BRG1 (Abcam, ab110641), b-Tubulin (Cell Signaling, 2128), NRF2 (Abcam, ab62352), H3K27me3 (Thermofisher, MA511198), H3 (Cell Signaling, 9715), KEAP1 (Cell Signaling, 4678), TKT (Cell Signaling, 64414), AKR1B10 (Abcam, ab192865), GCLC (Cell Signaling, 48005) and POPDC3(Novus Biologicals, NBP159476).

Techniques: Activation Assay, Activity Assay, Imaging, Expressing, Mutagenesis

Journal: Cell reports

Article Title: CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

doi: 10.1016/j.celrep.2024.114345

Figure Lengend Snippet: Figure 5. The SWI/SNF complex binds to NRF2 sites at enhancers and promoters to increase chromatin accessibility and gene expression (A and G) Pile-up plots of normalized mean read counts for the indicated antibodies from Cut&Run experiments (n = 2 biological replicates) generated using consensus sites. (B, C, H, I) Venn diagram of gene-level overlap between the indicated factors. The number of regions are indicated for each condition. For (B), p = 1e1,037 for overlap between NRF2 and BRM, p = 1e1,291 for overlap between BRM, NRF2, and SMARCC1, and p = 1e2,064 for overlap between BRM and SMARCC1. For (H), p = 1e504 for overlap between NRF2 and BRG1, p = 1e145 for overlap between BRG1, NRF2, and SMARCC1, and p = 1e135 for overlap between BRG1 and SMARCC1. For (B) and (H), p values were determined using hypergeometric distribution. (D and J) Genome browser view of BRM Cut&Run (D) or BRG1 Cut&Run (J) for the indicated antibodies at TKT, a NRF2 target gene.

Article Snippet: The following antibodies were used in this study: BRM (Cell Signaling, 11966), BRG1 (Abcam, ab110641), b-Tubulin (Cell Signaling, 2128), NRF2 (Abcam, ab62352), H3K27me3 (Thermofisher, MA511198), H3 (Cell Signaling, 9715), KEAP1 (Cell Signaling, 4678), TKT (Cell Signaling, 64414), AKR1B10 (Abcam, ab192865), GCLC (Cell Signaling, 48005) and POPDC3(Novus Biologicals, NBP159476).

Techniques: Gene Expression, Generated

Journal: Cell reports

Article Title: CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

doi: 10.1016/j.celrep.2024.114345

Figure Lengend Snippet: Figure 6. The SWI/SNF complex enhances NRF2 binding to the chromatin (A) Representative western blot showing NRF2, TUBULIN, Histone H3, and BRG1 protein in the indicated cellular fractions in KP4 cells. (B) Bar graph quantifications (mean ± SEM, n = 3 biological replicates) showing relative chromatin to cytoplasmic ratio of NRF2 protein from 3 independent experiments. The chromatin to cytoplasmic ratio of NRF2 in LACZ-expressing cells was set to 1. p values were determined by a 1-way ANOVA test with Tukey’s correction for multiple comparisons. (C) Representative ML210 dose-response curves (mean ± SEM, n = 3 independently treated wells) of cells overexpressing BRM transduced with a sgRNA targeting NRF2 (sgNRF2) or a non-targeting sgRNA (sgNT). p values derived from a 2-way ANOVA with Tukey’s correction for multiple comparisons.

Article Snippet: The following antibodies were used in this study: BRM (Cell Signaling, 11966), BRG1 (Abcam, ab110641), b-Tubulin (Cell Signaling, 2128), NRF2 (Abcam, ab62352), H3K27me3 (Thermofisher, MA511198), H3 (Cell Signaling, 9715), KEAP1 (Cell Signaling, 4678), TKT (Cell Signaling, 64414), AKR1B10 (Abcam, ab192865), GCLC (Cell Signaling, 48005) and POPDC3(Novus Biologicals, NBP159476).

Techniques: Binding Assay, Western Blot, Expressing, Transduction, Derivative Assay

Journal: Cell reports

Article Title: CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

doi: 10.1016/j.celrep.2024.114345

Figure Lengend Snippet: Figure 6. The SWI/SNF complex enhances NRF2 binding to the chromatin (A) Representative western blot showing NRF2, TUBULIN, Histone H3, and BRG1 protein in the indicated cellular fractions in KP4 cells. (B) Bar graph quantifications (mean ± SEM, n = 3 biological replicates) showing relative chromatin to cytoplasmic ratio of NRF2 protein from 3 independent experiments. The chromatin to cytoplasmic ratio of NRF2 in LACZ-expressing cells was set to 1. p values were determined by a 1-way ANOVA test with Tukey’s correction for multiple comparisons. (C) Representative ML210 dose-response curves (mean ± SEM, n = 3 independently treated wells) of cells overexpressing BRM transduced with a sgRNA targeting NRF2 (sgNRF2) or a non-targeting sgRNA (sgNT). p values derived from a 2-way ANOVA with Tukey’s correction for multiple comparisons.

Article Snippet: The following antibodies were used in this study: BRM (Cell Signaling, 11966), BRG1 (Abcam, ab110641), b-Tubulin (Cell Signaling, 2128), NRF2 (Abcam, ab62352), H3K27me3 (Thermofisher, MA511198), H3 (Cell Signaling, 9715), KEAP1 (Cell Signaling, 4678), TKT (Cell Signaling, 64414), AKR1B10 (Abcam, ab192865), GCLC (Cell Signaling, 48005) and POPDC3(Novus Biologicals, NBP159476).

Techniques: Binding Assay, Western Blot, Expressing, Transduction, Derivative Assay