Figure 7.
Generation of ABT-199–resistant LOUCY cell line phenocopies BCL-2 independence and altered differentiation. (A) Scheme to generate LOUCY cells resistant to ABT-199. Parental LOUCY cells were exposed to either vehicle (DMSO) or ABT-199. Initially, LOUCY cells were treated at low dose of ABT-199 to reach a population of resistant clones that were capable of maintaining viability with continuous exposure to ABT-199 up to 2 μM. (B) BH3 profile of parental cells (CTRL) and the ABT-199–resistant clones (ABT-199R) (mean ± standard deviation [SD]; n = 3). Two-way analysis of variance with Dunnett’s multiple comparison for statistical analysis was performed. (C) Percentage of surviving cells after ABT-199 and WEHI-539 treatment, in parental and resistant cell lines, graphed is the dose–response curves, and subsequent 50% inhibitory concentration (IC50) values are listed (mean ± SD; n = 3). Two-way analysis of variance with Dunnett’s multiple comparison post hoc test. (D-E) Expression analysis of BCL-2 and BCL-XL proteins by western blot, the correspondent densitometry analysis (top panel) and quantitative real-time polymerase chain reaction analysis (bottom panel). β-actin used as loading control to normalize protein expression. The mRNA gene expression was normalized to GAPDH. (F) Representative flow cytometry plot of the mean florescence intensity (MFI) for surface CD3 expression (sCD3-APC) of parental CTRL and ABT-199R cells. sCD3 expression of parental CTRL and ABT-199R cells; data graphed are presented in MFI (n = 3; mean ± SD). (G) Representative flow cytometry plot of the MFI for CD1a-APC expression of parental CTRL and ABT-199R cells. CD1a expression of parental CTRL and ABT-199R cells; data graphed are presented in MFI (n = 3; mean ± SD). (H) GATA3 (n = 3; mean ± SD), TAL1 (n = 1 mean ± SD of triplicate), and LMO2 (n = 3; mean ± SD) mRNA levels in parental CTRL and ABT-199R cells measured by quantitative reverse transcription polymerase chain reaction. The mRNA gene expression was normalized to RP18S. Paired Student t test statistical analysis was conducted to determine the P values. *P ≤ .05, **P ≤ .01, ***P ≤ .001.

Generation of ABT-199–resistant LOUCY cell line phenocopies BCL-2 independence and altered differentiation. (A) Scheme to generate LOUCY cells resistant to ABT-199. Parental LOUCY cells were exposed to either vehicle (DMSO) or ABT-199. Initially, LOUCY cells were treated at low dose of ABT-199 to reach a population of resistant clones that were capable of maintaining viability with continuous exposure to ABT-199 up to 2 μM. (B) BH3 profile of parental cells (CTRL) and the ABT-199–resistant clones (ABT-199R) (mean ± standard deviation [SD]; n = 3). Two-way analysis of variance with Dunnett’s multiple comparison for statistical analysis was performed. (C) Percentage of surviving cells after ABT-199 and WEHI-539 treatment, in parental and resistant cell lines, graphed is the dose–response curves, and subsequent 50% inhibitory concentration (IC50) values are listed (mean ± SD; n = 3). Two-way analysis of variance with Dunnett’s multiple comparison post hoc test. (D-E) Expression analysis of BCL-2 and BCL-XL proteins by western blot, the correspondent densitometry analysis (top panel) and quantitative real-time polymerase chain reaction analysis (bottom panel). β-actin used as loading control to normalize protein expression. The mRNA gene expression was normalized to GAPDH. (F) Representative flow cytometry plot of the mean florescence intensity (MFI) for surface CD3 expression (sCD3-APC) of parental CTRL and ABT-199R cells. sCD3 expression of parental CTRL and ABT-199R cells; data graphed are presented in MFI (n = 3; mean ± SD). (G) Representative flow cytometry plot of the MFI for CD1a-APC expression of parental CTRL and ABT-199R cells. CD1a expression of parental CTRL and ABT-199R cells; data graphed are presented in MFI (n = 3; mean ± SD). (H) GATA3 (n = 3; mean ± SD), TAL1 (n = 1 mean ± SD of triplicate), and LMO2 (n = 3; mean ± SD) mRNA levels in parental CTRL and ABT-199R cells measured by quantitative reverse transcription polymerase chain reaction. The mRNA gene expression was normalized to RP18S. Paired Student t test statistical analysis was conducted to determine the P values. *P ≤ .05, **P ≤ .01, ***P ≤ .001.

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