Fig. 1: MPL S505C is found together with W515R in ET/MF and enhances autonomous activation of W515R and W515L in vitro.

A Patient Sanger sequencing demonstrating heterozygous S505C and W515R mutations. B Dual luciferase assay for STAT5 transcription activity of MPL variants with and without TPO stimulation. HEK 293T cells were transiently transfected with plasmids for the expression of HA-tagged MPL WT or variants, or pMX-IRES-GFP empty vector (EV), and treated or not with 10 ng/mL recombinant human TPO. Relative luminescence was determined using a Dual Luciferase Reporter Assay system (Promega). Mean ± SD is shown for n = 3 independent experiments performed in triplicate, normalized to relative luciferase activity with overexpression of MPL WT in the absence of cytokine stimulation. *: p < 0.05, **: p < 0.01, ***: p < 0.001, by Student’s t-test. C Factor-free proliferation assay in Ba/F3 cells. BaF3 cells were transfected with viral supernatants from HEK293T cells to express HA-tagged MPL WT or variants. Stable lines over expressing MPL were selected by flow cytometry-based on cell surface HA expression. Stable cell lines were then cultured for seven days in factor free conditions. Cell viability was determined using the Cell Titer Glo assay (Promega) on days 0, 3, 5, and 7. D. Cross-linking of MPL variants in the absence of TPO. A truncated form of MPL, lacking cytoplasmic region (1-560AA) was expressed in CHO cells. 48 h post transfection, cells were treated or not with 100 µM oPDM in DMSO for 10 min. Lysates were examined by Western blot for HA-MPL expression with an antibody directed towards HA (C29F4, Cell Signaling). * non-specific band. All cell lines used were routinely tested for mycoplasma contamination.