Fig. 6: JAK2 downregulation increases NRAS mutated cells oncogenic potential through cellular release from oncogene-induced senescence.
From: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms
ANRAS and JAK2 expression level qRT-PCR in Empty or NRASQ61K HEL and UKE-1 cells. Error bars represent mean of n = 3 (HEL) and n = 4 (UKE-1) biological replicates ± SD. B Western blot for indicated proteins, in Empty or NRASQ61K HEL and UKE-1. The experiment was performed twice with similar results. C, D Gene Set Enrichment Analysis (GSEA) for the AML dataset Beat_AML_v2, across the collection of MSigDB_v7.4 Hallmark (50) and additional RAS/MAPK (24) and cell cycle (11) gene sets. Data represented as volcano plots (C) of –log10(p-value + 0.0001) versus the Normalized Enrichment Score (NES) for each gene set. NES using Kolmogorov-Smirnov enrichment test. Gene sets related to RAS/MAPK and Cell Cycle highlighted in red and blue respectively. Gray dots indicate all other. Representative GSEA plots (D) for gene sets related to RAS/MAPK and Cell Cycle. E Heatmaps depicting relative gene expression changes for leading edge genes of the top RAS/MAPK and Cell Cycle enriched gene sets. Depleted and enriched genes are respectively in blue and red. Row-normalized data. F Propidium Iodide cell cycle analysis of Empty or NRASQ61K HEL and UKE-1 cells after 11 or 9 days of ruxolitinib (3 and 15 µM, respectively). Cells in sub G1 not represented. Mean of n = 2 biological replicates. G EdU incorporation of Empty or NRASQ61K HEL and UKE-1 cells after 12 days of ruxolitinib (3 and 15 µM, respectively). Error bars represent the mean of n = 3 biological replicates ± SEM. H, I Beta-galactosidase staining for Empty or NRASQ61K HEL and UKE-1 cells after 6 days of ruxolitinib (3 and 15 µM, respectively). Quantification (H) and representative images (I). Error bars represent mean of n = 2 (HEL) and n = 3 (UKE-1) biological replicates ± SD. J Proliferation curves of HEL cells expressing Empty, NrasQ61K, JAK2V617F or NrasQ61K and JAK2V617F vectors. Error bars represent mean ± SD of n = 10 (day 0), n = 8 (days 3, 6), n = 4 (day 9) biological replicates. Statistical significance using two-tailed Welch’s t-test (A, F–H, J). P-values are reported in the figure. Source data provided as a Source Data file.
