Figure 2.
Increased SGK1 expression promotes cell growth and AKT independence. (A) CRISPR-edited control or splice-mutant Defauw clones were labeled with GFP and pooled for competitive growth. The relative proportion of control and mutant clones is shown over a 40-day time course. Data reflect 3 separate pairings of 3 WT and 3 mutant clones. Significance was calculated by paired Student t test. *P < .05. (B) Primary human germinal center B cells were immortalized with BCL6-2A-BCL2 and then transduced with cDNAs encoding canonical or truncated SGK1 isoforms. Constructs encoding SGK1 isoforms are named by the position of the initiating methionine. The frequency of cells transduced with each SGK1 isoform was quantified by flow cytometry at intervals of >30 days and normalized to day 4. (C) Cell viability measured by CellTiter-Glo after a 5-day treatment with the AKT inhibitor AZD5363 in BJAB cells transduced with control, canonical (M1) or N-terminal truncated (M28, M32, and M69) SGK1 isoforms. The figure shows an average of 4 independently conducted experiments; error bars represent standard error of the mean (SEM). (D) BJAB cells were transduced with the indicated canonical or truncated SGK1 isoforms, with or without the K127N kinase-dead (KD) mutation. Apoptosis was quantified by intracellular staining for caspase-3 and cleaved PARP 3 days after treatment with 500 nM AZD5363. Data show average and SEM of 3 separate experiments. Significance compared with the empty vector control was calculated by analysis of variance (ANOVA) and Dunnett’s multiple comparisons test. ***P < .001. (E) Immunoblot stained for phospho- and total GSK3B. Lysates are from BJAB cells stably transduced with the indicated canonical, WT (M1), or N-terminal truncated (M28, M32, and M69) SGK1 isoforms and treated with AZD5363 (500 nM) or dimethyl sulfoxide (DMSO) for 24 hours. Data are representative of 3 independent experiments. (F) Defauw cells were treated with the indicated concentration of AZD5363 and the SGK1 inhibitor GSK650394, either individually or in combination. Cell viability was measured by CellTiter-Glo. Data show average and SEM of 3 separate experiments. Significance compared with DMSO control was calculated by ANOVA and Dunnett’s multiple comparisons test. **P < .01.

Increased SGK1 expression promotes cell growth and AKT independence. (A) CRISPR-edited control or splice-mutant Defauw clones were labeled with GFP and pooled for competitive growth. The relative proportion of control and mutant clones is shown over a 40-day time course. Data reflect 3 separate pairings of 3 WT and 3 mutant clones. Significance was calculated by paired Student t test. *P < .05. (B) Primary human germinal center B cells were immortalized with BCL6-2A-BCL2 and then transduced with cDNAs encoding canonical or truncated SGK1 isoforms. Constructs encoding SGK1 isoforms are named by the position of the initiating methionine. The frequency of cells transduced with each SGK1 isoform was quantified by flow cytometry at intervals of >30 days and normalized to day 4. (C) Cell viability measured by CellTiter-Glo after a 5-day treatment with the AKT inhibitor AZD5363 in BJAB cells transduced with control, canonical (M1) or N-terminal truncated (M28, M32, and M69) SGK1 isoforms. The figure shows an average of 4 independently conducted experiments; error bars represent standard error of the mean (SEM). (D) BJAB cells were transduced with the indicated canonical or truncated SGK1 isoforms, with or without the K127N kinase-dead (KD) mutation. Apoptosis was quantified by intracellular staining for caspase-3 and cleaved PARP 3 days after treatment with 500 nM AZD5363. Data show average and SEM of 3 separate experiments. Significance compared with the empty vector control was calculated by analysis of variance (ANOVA) and Dunnett’s multiple comparisons test. ***P < .001. (E) Immunoblot stained for phospho- and total GSK3B. Lysates are from BJAB cells stably transduced with the indicated canonical, WT (M1), or N-terminal truncated (M28, M32, and M69) SGK1 isoforms and treated with AZD5363 (500 nM) or dimethyl sulfoxide (DMSO) for 24 hours. Data are representative of 3 independent experiments. (F) Defauw cells were treated with the indicated concentration of AZD5363 and the SGK1 inhibitor GSK650394, either individually or in combination. Cell viability was measured by CellTiter-Glo. Data show average and SEM of 3 separate experiments. Significance compared with DMSO control was calculated by ANOVA and Dunnett’s multiple comparisons test. **P < .01.

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