plko rb1 shrna19  (Addgene inc)

Journal: Nature communications

Article Title: GLUT1 inhibition blocks growth of RB1-positive triple negative breast cancer.

doi: 10.1038/s41467-020-18020-8

Figure Lengend Snippet: Fig. 1 Growth of a subset of TNBC relies on GLUT1 activity. SLC2A1 gene expression in the a TCGA breast cancer datasets, b METABRIC breast cancer datasets, and c Princess Margaret Hospital PDXs datasets (PM-PDXs). According to PAM50 classification, the cohorts were designated as basal and non- basal subtypes. Gene expression is reported as log2(TPM + 0.001). The number of patients (n) per group is indicated. Wilcoxon rank sum test. *p < 0.05; ****p < 0.0001. d Heatmap of mean IC50 values for the indicated 21 breast cancer cell lines. n = 4, mean ± s.d. e Representative immunoblots showing the siRNA knockdown of GLUT1 or luciferase control in the BAY-876-sensitive lines (HCC1806 and Hs 578T) and BAY-876-resistant lines (MDA-MB-436 MDA-MB-468). Relative band intensities shown below blots. f Normalized cell confluency of GLUT1 knockdown cells or siRNA luciferase control cells for the indicated time post siRNA transduction. Cell confluency is normalized to T0 time point. n = 4, mean ± s.d. Two-way ANOVA. *p < 0.05; **p < 0.01; n.s. not significant. g Cell growth of TNBC lines cultured in complete DMEM medium with or without glucose deprivation for 5 days. n = 4, mean ± s.d. Two- way ANOVA. ****p < 0.0001. h Flow cytometry cell cycle analysis for indicated cells cultured with or without BAY-876 treated for 72 h. n = 3, mean ± s.d. Two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant. i Representative images of caspase 3/7 staining. The scale bar represents 300 µM. j Apoptotic cell counts of BAY-876 treated for 3 days by caspase 3/7 staining. n = 3, mean ± s.d. Two-way ANOVA. ***p < 0.001; ****p < 0.0001; n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: Two independent shRNA vectors targeting RB1 were obtained from Addgene (Addgene ID: 25640 and 25641).

Techniques: Activity Assay, Gene Expression, Western Blot, Knockdown, Luciferase, Control, Transduction, Cell Culture, Flow Cytometry, Cell Cycle Assay, Staining

Journal: Nature communications

Article Title: GLUT1 inhibition blocks growth of RB1-positive triple negative breast cancer.

doi: 10.1038/s41467-020-18020-8

Figure Lengend Snippet: Fig. 3 RB1 protein level discriminates response to GLUT1 inhibition. a Volcano plot of log2 fold change for all genes significantly upregulated (red; left) in sensitive lines or in resistant lines (blue; right). b Top enriched pathways in BAY-876-resistant lines compared to BAY-876-sensitive lines based on GSEA. c Heatmap of pathways correlated with OXPHOS revealed by GSEA of TCGA RNA-sequencing data. Clusters significantly related to OXPHOS are zoomed at the bottom. Pearson’s correlation coefficients between Log2 normalized protein expression data and response of BAY-876 showing significantly associated proteins with sensitive lines (red; left) or resistant lines (blue; right) based on the dataset from d MD-Anderson Cancer Center and e Princess Margaret Cancer Center (PMCC). f Representative western blot showing the variable RB1 expression levels in 17 TNBC lines. β-tubulin as a loading control. g Correlation of RB1 protein levels and the IC50 of BAY-876. h Representative immunoblot showing MDA-MB-436 cells expressing RB1 or GFP control proteins. β-tubulin as a loading control. i ECAR and OCR values were measured for MDA-MB-436 cells expressing RB1 or GFP control. n = 4; mean ± s.d. Two-sided Student’s t test. *p < 0.05; ***p < 0.001. j Growth curves of MDA-MB-436 cells expressing RB1 or GFP control in the presence of indicated concentrations BAY-876 treatment for 5 days. n = 4; mean ± s.d. k Representative western blot showing HCC1806 cells transfected with shRB1 and shLUC. β-actin as a loading control. l ECAR and OCR values were measured for HCC1806 cells transfected with shRB1 and shLUC. n = 4; mean ± s.d. Two-way ANOVA. *p < 0.05; ***p < 0.001. m Growth curves of HCC1806 cells with control knockdown or RB1 knockdown in the presence of indicated concentrations BAY-876 treatment for 5 days. n = 4; mean ± s.d. n Western blot showing the variable RB1 expression levels in a panel of three TNBC patient-derived organoids. β-actin as loading control. o Cell viability assays of patient-derived organoids with indicated concentrations of BAY-876. n = 3; mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Two independent shRNA vectors targeting RB1 were obtained from Addgene (Addgene ID: 25640 and 25641).

Techniques: Inhibition, RNA Sequencing, Expressing, Western Blot, Control, Transfection, Knockdown, Derivative Assay