Figure 3.
GNAS deficiency reduces NR3C1 levels and results in dexamethasone resistance in mouse and human T-ALL cells. (A) qPCR analysis of Nr3c1 mRNA in mouse T-ALLs expressing NS or Gnas shRNAs. (B) NR3C1 protein levels in mouse T-ALL cells stably transduced with NS or Gnas-specific shRNAs (TRC056060). Protein levels of 3 separate Western blots quantifed by densitometric analysis. (C) Intracellular cAMP levels of 1 × 106 mouse T-ALL cells stably transduced with NS or Gnas shRNAs after vehicle or forskolin (100 nM) treatment for 30 minutes. (D-E) qPCR analysis of Nr3c1 mRNA (D) and NR3C1 protein levels (E) in mouse 1390 T-ALL cells treated with dexamethasone (100 nM) or forskolin (100 nM) for 6 hours. (F) Quantification of apoptotic cells after 48 hours of vehicle or forskolin treatment (10 or 100 nM) of mouse T-ALL 1390 cells expressing NS or a Gnas shRNA. (G) qPCR analysis of GNAS mRNA in the human T-ALL cell line DND41 stably transduced with NS or GNAS-specific shRNAs. (H) Intracellular cAMP levels in 2 × 106 DND41 cells transduced with NS or GNAS-specific shRNAs treated with vehicle or forskolin (10µM) for 30 minutes. (I) Relative viability measured by cell titer glo of DND41 cells transduced with NS or GNAS-specific shRNAs after treatment with increasing concentrations of forskolin (0-100 µM) for 72 hours. (J) NR3C1 and ERK1/2 protein levels in DND41 cells transduced with NS or GNAS-specific shRNAs. (K) Quantification of apoptotic DND41 cells transduced with NS or GNAS-specific shRNAs after vehicle or dexamethasone treatment (100 nM) for 72 hours. (L) Relative viability measured by cell titer glo of DND41 cells transduced with NS or GNAS-specific shRNAs after treatment with increasing concentrations of dexamethasone (0-10 µM) for 72 hours. Data are normalized to vehicle-treated cells. (M) qPCR analysis of NR3C1 mRNA levels in DND41 cells transduced with NS or GNAS shRNAs treated with vehicle, dexamethasone (1 µM), or forskolin (10 µM) for 6 hours. (N) qPCR analysis of NR3C1 target genes in DND41 cells transduced with NS or GNAS shRNAs and treated with vehicle or dexamethasone (1 µM) for 6 hours. (O) Relative viability measured by cell titer glo of NALM-6 cells transduced with NS or GNAS-specific shRNAs after treatment with increasing concentrations of dexamethasone (0-10 µM) for 72 hours. Data are normalized to vehicle-treated cells. (P) qPCR analysis of GNAS, NR3C1, BCL2L11, and TSC22D3 in NALM-6 cells transduced with NS or GNAS shRNAs treated with vehicle or dexamethasone (1 µM) for 6 hours. ALL results are averages of at least 3 independent experiments, and error bars represent SEM. *P < .05, **P < .01, ***P < .001.

GNAS deficiency reduces NR3C1 levels and results in dexamethasone resistance in mouse and human T-ALL cells. (A) qPCR analysis of Nr3c1 mRNA in mouse T-ALLs expressing NS or Gnas shRNAs. (B) NR3C1 protein levels in mouse T-ALL cells stably transduced with NS or Gnas-specific shRNAs (TRC056060). Protein levels of 3 separate Western blots quantifed by densitometric analysis. (C) Intracellular cAMP levels of 1 × 106 mouse T-ALL cells stably transduced with NS or Gnas shRNAs after vehicle or forskolin (100 nM) treatment for 30 minutes. (D-E) qPCR analysis of Nr3c1 mRNA (D) and NR3C1 protein levels (E) in mouse 1390 T-ALL cells treated with dexamethasone (100 nM) or forskolin (100 nM) for 6 hours. (F) Quantification of apoptotic cells after 48 hours of vehicle or forskolin treatment (10 or 100 nM) of mouse T-ALL 1390 cells expressing NS or a Gnas shRNA. (G) qPCR analysis of GNAS mRNA in the human T-ALL cell line DND41 stably transduced with NS or GNAS-specific shRNAs. (H) Intracellular cAMP levels in 2 × 106 DND41 cells transduced with NS or GNAS-specific shRNAs treated with vehicle or forskolin (10µM) for 30 minutes. (I) Relative viability measured by cell titer glo of DND41 cells transduced with NS or GNAS-specific shRNAs after treatment with increasing concentrations of forskolin (0-100 µM) for 72 hours. (J) NR3C1 and ERK1/2 protein levels in DND41 cells transduced with NS or GNAS-specific shRNAs. (K) Quantification of apoptotic DND41 cells transduced with NS or GNAS-specific shRNAs after vehicle or dexamethasone treatment (100 nM) for 72 hours. (L) Relative viability measured by cell titer glo of DND41 cells transduced with NS or GNAS-specific shRNAs after treatment with increasing concentrations of dexamethasone (0-10 µM) for 72 hours. Data are normalized to vehicle-treated cells. (M) qPCR analysis of NR3C1 mRNA levels in DND41 cells transduced with NS or GNAS shRNAs treated with vehicle, dexamethasone (1 µM), or forskolin (10 µM) for 6 hours. (N) qPCR analysis of NR3C1 target genes in DND41 cells transduced with NS or GNAS shRNAs and treated with vehicle or dexamethasone (1 µM) for 6 hours. (O) Relative viability measured by cell titer glo of NALM-6 cells transduced with NS or GNAS-specific shRNAs after treatment with increasing concentrations of dexamethasone (0-10 µM) for 72 hours. Data are normalized to vehicle-treated cells. (P) qPCR analysis of GNAS, NR3C1, BCL2L11, and TSC22D3 in NALM-6 cells transduced with NS or GNAS shRNAs treated with vehicle or dexamethasone (1 µM) for 6 hours. 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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