Extended Data Fig. 7: MEK-inhibition prevents YBX1 nuclear localization in Jak2-mutated cells.
From: Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms
a, Confocal images of YBX1 localization (Red) in Ba/F3 JAK2VF cells after treatment for 2 h with RUX (0.5 μM), MEK inhibitor (2 μM), the ERK inhibitor trametinib (Tram; 100 and 200 nM), RUX + Tram or DMSO (control). Cells were counterstained with DAPI. b, Bar plot shows quantification of nuclear YBX1 expression in Ba/F3 JAK2VF cells (n ≥ 3 independent imaging experiments, P value determined by Student’s t-test). DMSO (n = 67): min = 7.05; max = 27.27; whisker = [7.05–27.27]; median = [15.01], RUX (n = 77): min = 6.6; max = 35.8; whisker = [6.6-35.8]; median = [16.39], CGP57380 (n = 89): min = 3.2; max = 16.35; whisker = [3.2–16.35]; median = [8.35], Tram (n = 67): min = 2.44; max = 14.18; whisker = [2.44–14.18]; median = [12.01], Tram (n = 62): min = 2.5; max = 17.96; whisker = [2.5–17.96]; median = [12.01], Tram + RUX (n = 64): min = 3.44; max = 19.23; whisker = [3.44–19.23]; median = [13.04], MEK inhibitor + RUX (n = 68): min = 4.06 max = 14.68; whisker = [4.06–14.68]; median = [13.01]. c, Bubble plot showing the regulation of human YBX1 pS32 and pS36 phosphorylation in HEL cells treated with RUX (0.5 μM), CGP57380 (10 μM), Tram (500 nM and 2 μM) or DMSO (control) for 4 h in vitro. Phosphorylation status of MAPK and JAK2 is shown as successful inhibition of respective targets of the corresponding kinase inhibitors. n = 4 biological samples per group. Size and colour of bubbles are proportional to the z-scored log2 phosphosite intensity; significance assessed using multiple sample test. d, Confocal images of YBX1 localization (red) in HEL cells treated with inhibitors or DMSO for 2 h. Cells were counterstained with DAPI. n = 3 biological experiments. e, Bar plot shows quantification of nuclear YBX1 expression in HEL cells; P values were determined by Student’s t-test. DMSO (n = 44): min = 17.51; max = 34.56; whisker = [17.51–34.56];median = [24.30], RUX (n = 44): min = 13.04; max = 36.92; whisker = [13.04–36.92]; median = [23.9], MEK inhibitor (n = 41): min = 7.62; max = 15.36; whisker = [7.62-15.36]; median = [11.89], Tram (n = 48): min = 6.38; max = 19.03; whisker = [6.38-19.03]; median = [12.01], Tram + RUX (n = 55): min = 3.31; max = 19.19; whisker = [3.31-19.19]; median = [13.04], MEK inhibitor + RUX (n = 49): min = 4.32; max = 19.46; whisker = [4.32-19.46]; median = [13.01]. f, Immunoprecipitation of YBX1 from Ba/F3 JAK2VF cells expressing YBX1 phosphomutants. n = 3 independent experiments.
