Extended Data Fig. 9: Targeting Mknk1 deregulates ERK signalling in Jak2-mutated cells.
From: Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms
a, Unsupervised hierarchical clustering of significantly down regulated phosphosites (n = 2,390 sites) in human HEL cells following inactivation of YBX1 by two independent shRNAs compared to non-targeting control. n = 4 biological replicates. Heatmap represents z-scored and averaged log2 phosphosite intensity, significance by ANOVA test with permutation-based FDR < 0.01. b, Kinase-substrate motifs significantly downregulated in YBX1-targeted HEL cells are shown including Benjamini–Hochberg FDR value (–log10). c, ERK substrate motifs significantly downregulated and shared between Ybx1-targeted mouse and human JAK2VF cells. d, Western blot analysis of total protein abundance and phosphorylation of JAK2 downstream targets upon treatment with JAK inhibitor and/or genetic inactivation of YBX1 by RNAi. GAPDH used as loading control. Representative images from n = 3 independent experiments. e, f, Bar plots show the mean fluorescence intensity of pERK levels measured in human HEL (e; n = 3) and in patient JAK2-mutated cells (f; n = 4 independent biological replicates from 4 individual patients) following genetic inactivation of YBX1 by RNAi with or without drug treatment as indicated. Representative FACS plots shown in Fig. 4d. Data shown as mean ± s.d. and P value determined by two-tailed Student’s t-test. g, Western blot validation of Mknk1-targeting shRNAs in mouse Ba/F3 JAK2VF cells. h, Representative western blot analysis of pERK upon genetic inactivation of Mknk1 in Ba/F3 JAK2VF cells. n = 4 with comparable results. i, Growth curve of JAK2VF cells following lentiviral infection with shRNAs targeting Mknk1 or non-targeting control and treatment with increase doses of JAK inhibitor (1nM–10 μM RUX) measured by MTS assay. n = 4, each with 8 technical replicates. j, Percentage of apoptotic JAK2VF cells following lentiviral knockdown of Mknk1 (sh2, sh3 ± RUX 0.5 μM) or infection with non-targeting control (shSCR) (n = 4, two-tailed Student t-test, mean ± s.d.).
