Figure 1.
A genome-wide shRNA screen for dexamethasone resistance identify genes enriched in cAMP signaling. (A) Schematic of whole-genome survival-based shRNA screen performed in the mouse T-ALL cell line 1390. (B) Biological reproducibility of the relative changes in shRNA abundance between 2 independent preparations of shRNA MiSeq libraries. (C) Number of shRNAs identified by counts per million reads in MiSeq analysis. (D) shRNA rank by average counts per million determined by MiSeq. shRNAs to the glucocorticoid receptor (Nr3c1) are highlighted in red. (E) Annexin V/7AAD staining of mouse T-ALL 1390 cells stably expressing nonsilencing (NS) or Nr3c1 shRNAs treated with vehicle or dexamethasone (50 nM) for 48 hours. (F) Quantitative PCR (qPCR) analysis of Nr3c1 mRNA in mouse T-ALL cells transduced with Nr3c1-specific shRNAs or an NS shRNA after dexamethasone (100 nM) treatment of 6 hours. (G) NR3C1 protein levels in mouse T-ALL 1390 cells expressing NS or Nr3c1 shRNAs treated with vehicle or dexamethasone (100 nM) for 6 hours. (H) Venn diagram showing genes identified in shRNA screen that interact with NR3C1, have tumor suppressor function, been previously implicated in glucocorticoid resistance, and are mutated in ALL patients. (I) Validation of selected shRNAs identified in the shRNA screen using MTS assay in mouse T-ALL cell line 1390 after dexamethasone (0-1 μM) treatment of 48 hours. All data were normalized to vehicle-treated cells. (J) Volcano plot showing genes identified in shRNA screen cross referenced with genes differentially expressed in microarray of paired ALL patient samples at the time of diagnosis or at relapse. (K) Venn diagram of genes identified in shRNA screen and decreased in microarray tables of relapse ALL patients (P < .05; N = 49 paired patient samples). (L) KEGG pathway analysis performed in the Database for Annotation, Visualization and Integrated Discovery (DAVID) on genes identified in shRNA screen and genes significantly downregulated at the time of relapse (GSE28460, or in GC-resistant patient samples (GSE66702, GSE66705). (M) Venn diagram of cAMP genes identified in shRNA screen and decreased in relapsed patient (relapsed genes, right circle) or GC resistant patient samples (resistance genes, left circle). (N) Expression levels of cAMP genes in 49 paired samples at diagnosis and on relapse as determined by microarray identified in shRNA screen (GSE28460). Paired t test values are shown, and connecting lines represent paired samples. All results are averages of at least 3 independent experiments, and error bars represent SEM. *P < .05, **P < .01, ***P < .001.

A genome-wide shRNA screen for dexamethasone resistance identify genes enriched in cAMP signaling. (A) Schematic of whole-genome survival-based shRNA screen performed in the mouse T-ALL cell line 1390. (B) Biological reproducibility of the relative changes in shRNA abundance between 2 independent preparations of shRNA MiSeq libraries. (C) Number of shRNAs identified by counts per million reads in MiSeq analysis. (D) shRNA rank by average counts per million determined by MiSeq. shRNAs to the glucocorticoid receptor (Nr3c1) are highlighted in red. (E) Annexin V/7AAD staining of mouse T-ALL 1390 cells stably expressing nonsilencing (NS) or Nr3c1 shRNAs treated with vehicle or dexamethasone (50 nM) for 48 hours. (F) Quantitative PCR (qPCR) analysis of Nr3c1 mRNA in mouse T-ALL cells transduced with Nr3c1-specific shRNAs or an NS shRNA after dexamethasone (100 nM) treatment of 6 hours. (G) NR3C1 protein levels in mouse T-ALL 1390 cells expressing NS or Nr3c1 shRNAs treated with vehicle or dexamethasone (100 nM) for 6 hours. (H) Venn diagram showing genes identified in shRNA screen that interact with NR3C1, have tumor suppressor function, been previously implicated in glucocorticoid resistance, and are mutated in ALL patients. (I) Validation of selected shRNAs identified in the shRNA screen using MTS assay in mouse T-ALL cell line 1390 after dexamethasone (0-1 μM) treatment of 48 hours. All data were normalized to vehicle-treated cells. (J) Volcano plot showing genes identified in shRNA screen cross referenced with genes differentially expressed in microarray of paired ALL patient samples at the time of diagnosis or at relapse. (K) Venn diagram of genes identified in shRNA screen and decreased in microarray tables of relapse ALL patients (P < .05; N = 49 paired patient samples). (L) KEGG pathway analysis performed in the Database for Annotation, Visualization and Integrated Discovery (DAVID) on genes identified in shRNA screen and genes significantly downregulated at the time of relapse (GSE28460, or in GC-resistant patient samples (GSE66702, GSE66705). (M) Venn diagram of cAMP genes identified in shRNA screen and decreased in relapsed patient (relapsed genes, right circle) or GC resistant patient samples (resistance genes, left circle). (N) Expression levels of cAMP genes in 49 paired samples at diagnosis and on relapse as determined by microarray identified in shRNA screen (GSE28460). Paired t test values are shown, and connecting lines represent paired samples. All results are averages of at least 3 independent experiments, and error bars represent SEM. *P < .05, **P < .01, ***P < .001.

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