Figure 1.
MPLR464G is only weakly expressed on the cell surface and induces very weak signaling compared with MPLWT. (A) Flow cytometric analyses of MPL expression on platelets from 1 patient homozygous for MPLR464G (II.2), her heterozygous mother (I.2), and 1 healthy control (CTR). (B) Western blot analysis of TPO-induced signaling in UT-7 cells without MPL expression (UT-7), overexpressing the WT form of MPL (UT-7 MPLWT), and mutant MPL (UT-7 MPLR464G). Cells were starved overnight and stimulated with 10 ng/mL TPO for 10 or 30 minutes. No signal is detected in parental UT-7 cells, and a weak ERK signal is detected in the presence of MPLR464G in UT-7 cells. (C) Flow cytometric analyses of MPL expression on the surface of Ba/F3 cells overexpressing MPLWT and MPLR464G. (D) Western blot analysis of the mature (85 kDa) and immature (80 kDa) forms of MPL. The mature form of MPL that is resistant to endoglycosidase H (Endo H) digestion and able to reach the cell membrane is not detectable in UT-7 cells overexpressing MPLR464G. (E) Immunofluorescence staining for the expression of MPL and CALR in UT-7 cells overexpressing MPLWT or MPLR464G. The orange arrows indicate MPL colocalization with CALR in the ER, and the white arrows indicate diffuse cytoplasmic/cell-surface MPL expression. Scale bars, 30 μm. An anti-MPL coupled with phycoerythrin (PE; A,C) and uncoupled anti-MPL antibodies (D-E) were used. (F-G) Proliferation curves and western blot analysis of signaling of UT-7 cells overexpressing MPLWT and MPLR464G in presence of TPO (10 ng/mL), ELT (2 μg/mL), TPO (10 ng/mL) + ELT (2 μg/mL), or without cytokines (no cytokines). (F) 5 × 104 cells were plated in triplicate in 24-well plates and counted every day for 4 days. The counts were reported to the condition of UT-7 MPLWT+TPO (in blue color). Shown are averages of 3 independent experiments at day 4, each performed in triplicate ± standard error of the mean (error bars). *P < .05; ns, not significant; Mann-Whitney unpaired, nonparametric 1-tailed test. (G) Cells were starved overnight and stimulated for 10 minutes. A weak ERK signaling is detected in the presence of MPLR464G in all 3 conditions (TPO, ELT, and TPO + ELT). No signaling is detected in nonstimulated (NS) cells. **P < .01; ***P < .005; ****P < .001.

MPLR464G is only weakly expressed on the cell surface and induces very weak signaling compared with MPLWT. (A) Flow cytometric analyses of MPL expression on platelets from 1 patient homozygous for MPLR464G (II.2), her heterozygous mother (I.2), and 1 healthy control (CTR). (B) Western blot analysis of TPO-induced signaling in UT-7 cells without MPL expression (UT-7), overexpressing the WT form of MPL (UT-7 MPLWT), and mutant MPL (UT-7 MPLR464G). Cells were starved overnight and stimulated with 10 ng/mL TPO for 10 or 30 minutes. No signal is detected in parental UT-7 cells, and a weak ERK signal is detected in the presence of MPLR464G in UT-7 cells. (C) Flow cytometric analyses of MPL expression on the surface of Ba/F3 cells overexpressing MPLWT and MPLR464G. (D) Western blot analysis of the mature (85 kDa) and immature (80 kDa) forms of MPL. The mature form of MPL that is resistant to endoglycosidase H (Endo H) digestion and able to reach the cell membrane is not detectable in UT-7 cells overexpressing MPLR464G. (E) Immunofluorescence staining for the expression of MPL and CALR in UT-7 cells overexpressing MPLWT or MPLR464G. The orange arrows indicate MPL colocalization with CALR in the ER, and the white arrows indicate diffuse cytoplasmic/cell-surface MPL expression. Scale bars, 30 μm. An anti-MPL coupled with phycoerythrin (PE; A,C) and uncoupled anti-MPL antibodies (D-E) were used. (F-G) Proliferation curves and western blot analysis of signaling of UT-7 cells overexpressing MPLWT and MPLR464G in presence of TPO (10 ng/mL), ELT (2 μg/mL), TPO (10 ng/mL) + ELT (2 μg/mL), or without cytokines (no cytokines). (F) 5 × 104 cells were plated in triplicate in 24-well plates and counted every day for 4 days. The counts were reported to the condition of UT-7 MPLWT+TPO (in blue color). Shown are averages of 3 independent experiments at day 4, each performed in triplicate ± standard error of the mean (error bars). *P < .05; ns, not significant; Mann-Whitney unpaired, nonparametric 1-tailed test. (G) Cells were starved overnight and stimulated for 10 minutes. A weak ERK signaling is detected in the presence of MPLR464G in all 3 conditions (TPO, ELT, and TPO + ELT). No signaling is detected in nonstimulated (NS) cells. **P < .01; ***P < .005; ****P < .001.

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