n-terminal his tev-tagged brd9 bd  (Addgene inc)

Journal: Scientific Reports

Article Title: SMARCA2-deficiency confers sensitivity to targeted inhibition of SMARCA4 in esophageal squamous cell carcinoma cell lines

doi: 10.1038/s41598-019-48152-x

Figure Lengend Snippet: Selective dependency of SMARCA2-deficient ESCC models on SMARCA4. ( A ) Time-resolved CRISPR-Cas9 depletion studies. Fold depletion of sgRNA- and GFP-co-expressing cells relative to day 3 (d3) post-transduction was assessed by flow cytometry analysis in SMARCA2-proficient (blue) and SMARCA2-deficient (red) cell lines over 28 days. Three individual sgRNAs were directed against the DEAD-like helicase superfamily (DEXDc) domain and the bromodomain (BD), respectively. Non-targeting (Neg. Contr.) and cell essential gene (POLR2A) targeting sgRNAs were included as negative and positive controls. Data are represented as mean ± SD of three independent experiments. ( B ) Heatmap summary of studies shown in (A) normalized to depletion of POLR2A-directed sgRNAs at day 28 (d28) and capillary Western immunoassay for SMARCA2 and SMARCA4 of ESCC cell lines shown in (A). GAPDH expression was used to monitor equal loading.

Article Snippet: Figure 5 Pharmacological targeting of SMARCA4 impairs proliferation of SMARCA2-deficient ESCC KYSE-30 cells. ( A ) Schematic representation of siRNA/sgRNA-resistant SMARCA4 variant with substituted BRD9 bromodomain (SMARCA4 res -BD BRD9 ).

Techniques: CRISPR, Expressing, Transduction, Flow Cytometry, Western Blot

Journal: Scientific Reports

Article Title: SMARCA2-deficiency confers sensitivity to targeted inhibition of SMARCA4 in esophageal squamous cell carcinoma cell lines

doi: 10.1038/s41598-019-48152-x

Figure Lengend Snippet: SMARCA4 dependency in SMARCA2-deficient ESCC cell lines is linked to its ATPase function. ( A ) Schematic representation of SMARCA4 domain structure. Location of ATP-binding- and bromodomain (BD)-inactivating mutations in siRNA/sgRNA-resistant SMARCA4 (SMARCA4 res ) is indicated. DEXDc, DEAD-like helicases superfamily; BD, bromodomain. ( B ) SMARCA2-deficient KYSE-510 cells were stably transduced with wild-type or mutant forms of SMARCA4 res . Transgene expression was monitored by capillary Western immunoassay in presence of siRNA-mediated knockdown of endogenous SMARCA4. Lysates were prepared 72 h post siRNA transfection. GAPDH expression was used to monitor equal loading. ( C ) Time-resolved CRISPR-Cas9 depletion studies in SMARCA4 res expressing KYSE-510 cells. Fold depletion of sgRNA- and GFP-co-expressing cells relative to day 3 (d3) post-transduction was assessed by flow cytometry analysis over 28 days (n = 1 experimental replicate).

Article Snippet: Figure 5 Pharmacological targeting of SMARCA4 impairs proliferation of SMARCA2-deficient ESCC KYSE-30 cells. ( A ) Schematic representation of siRNA/sgRNA-resistant SMARCA4 variant with substituted BRD9 bromodomain (SMARCA4 res -BD BRD9 ).

Techniques: Binding Assay, Stable Transfection, Transduction, Mutagenesis, Expressing, Western Blot, Knockdown, Transfection, CRISPR, Flow Cytometry

Journal: Scientific Reports

Article Title: SMARCA2-deficiency confers sensitivity to targeted inhibition of SMARCA4 in esophageal squamous cell carcinoma cell lines

doi: 10.1038/s41598-019-48152-x

Figure Lengend Snippet: SMARCA2 and SMARCA4 display an acute synthetic lethal interaction in ESCC cells lines. ( A ) SMARCA2 reconstitution in SMARCA2-deficient KYSE-510 cells alleviates SMARCA4 dependency. Location of ATP-binding- and bromodomain (BD)-inactivating mutations in SMARCA2 (designated as SMARCA2 ect ) is indicated. DEXDc, DEAD-like helicases superfamily; BD, bromodomain. Time-resolved CRISPR-Cas9 depletion studies in SMARCA2 ect expressing KYSE-510 cells. Fold depletion of sgRNA- and GFP-co-expressing cells relative to day 3 (d3) post-transduction was assessed by flow cytometry analysis over 28 days (n = 1 experimental replicate). Expression of SMARCA2 ect variants was determined using capillary Western immunoassay. GAPDH expression was used to monitor equal loading. ( B ) SMARCA2 inactivation elicits dependency on SMARCA4. Time-resolved CRISPR-Cas9 depletion studies in SMARCA2-proficient and -deficient (KO) KYSE-450 monoclonal cell lines. Fold depletion of sgRNA- and GFP-co-expressing cells relative to day 3 (d3) post-transduction was assessed by flow cytometry analysis over 28 days (n = 1 experimental replicate). Knock-out of SMARCA2 was confirmed using capillary Western immunoassay. GAPDH expression was used to monitor equal loading.

Article Snippet: Figure 5 Pharmacological targeting of SMARCA4 impairs proliferation of SMARCA2-deficient ESCC KYSE-30 cells. ( A ) Schematic representation of siRNA/sgRNA-resistant SMARCA4 variant with substituted BRD9 bromodomain (SMARCA4 res -BD BRD9 ).

Techniques: Binding Assay, CRISPR, Expressing, Transduction, Flow Cytometry, Western Blot, Knock-Out

Journal: Scientific Reports

Article Title: SMARCA2-deficiency confers sensitivity to targeted inhibition of SMARCA4 in esophageal squamous cell carcinoma cell lines

doi: 10.1038/s41598-019-48152-x

Figure Lengend Snippet: Pharmacological targeting of SMARCA4 impairs proliferation of SMARCA2-deficient ESCC KYSE-30 cells. ( A ) Schematic representation of siRNA/sgRNA-resistant SMARCA4 variant with substituted BRD9 bromodomain (SMARCA4 res -BD BRD9 ). KYSE-30 cells were stably transduced with SMARCA4 res -BD BRD9 followed by knock-out of endogenous SMARCA4 and generation of a monoclonal cell line (KYSE-30-SMARCA4 res -BD BRD9 ). ( B ) Capillary Western immunoassay for SMARCA4 in parental KYSE-30 and KYSE-30-SMARCA4 res -BD BRD9 upon dBRD9 treatment for 4 h with the indicated doses. GAPDH expression was used to monitor equal loading. Quantification of SMARCA4 expression (lower panel) is represented as mean ± SD of four independent experiments. ( C ) Cell viability assay. Parental KYSE-30 cells and KYSE-30-SMARCA4 res -BD BRD9 were treated as indicated with dBRD9 and cell viability was determined ten days post treatment. Data are represented as mean ± SD of four independent experiments.

Article Snippet: Figure 5 Pharmacological targeting of SMARCA4 impairs proliferation of SMARCA2-deficient ESCC KYSE-30 cells. ( A ) Schematic representation of siRNA/sgRNA-resistant SMARCA4 variant with substituted BRD9 bromodomain (SMARCA4 res -BD BRD9 ).

Techniques: Variant Assay, Stable Transfection, Transduction, Knock-Out, Western Blot, Expressing, Viability Assay