Page 119 - 2021_12-Haematologica-web

MCL-1 in human hematopoiesis
AB
C
DE
FG
Figure 1. MCL-1 inhibition selectively sensitizes human CD34+ cells to endoplasmic reticulum stress. (A) HEK293T cells were transfected with plasmids expressing shRNA specific for Luciferase (shLuci) or human MCL-1 (shM#3 or shM#4). MCL-1 mRNA expression was determined in sorted GFP+ cells and normalized to the 36B4 reference gene. Bars represent mean ± standard error of mean (SEM); n=5 from five independent experiments. Human cord blood-derived CD34+ cells were trans- duced with the corresponding lentiviruses. GFP+ cells were sorted 24 h after transduction, and knockdown efficiency of MCL-1 was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). mRNA expression was normalized to 18S expression. Bars represent mean ± SEM; n=2-4 from four independent experiments. Mann-Whitney test: *P<0.05. (B, C) MCL-1 protein levels were determined in HEK293T (B) and CD34+ (C) GFP+ cells. (D) Measurements of apoptosis in CD34+ cells 24 h after lentiviral transduction revealed that 14-19% of cells undergo apoptosis early after MCL-1 depletion. Bars represent mean ± SEM; n=4 from four independent experiments. (E, F) Transduced CD34+ cells (transduction efficiency 45-65%) were cultured either under optimal conditions (cytokines and 10% serum) or under conditions of stress; in the presence of serum but deprived of cytokines, etoposide (0.5 mg/mL), taxol (0.125 mg/mL), tunicamycin I and II (0.5 and 1 mg/mL, respectively), thapsigargin (3 mM) or brefeldin A (BFA; 0.5 mg/mL). Apoptosis in GFP+ cells was determined by flow cytometry using annexin V and 7-AAD staining 24 h (E) and 48 h (F) later. Bars represent mean ± SEM; n=3-8 from eight independent experiments. Mann-Whitney test: *P<0.05, **P<0.01, ***P<0.001. (G) RNA was isolated from CD34+ cells treated with increasing concentrations of tunicamycin and used for qRT-PCR. MCL-1 mRNA expression was normalized to 18S expression. Bars represent mean ± SEM; n=4 from four independent experiments. Mann-Whitney test: *P<0.05.
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