Fig. 3

Expression of Xiap and Mapk8 is downregulated in JAK2^V617F+ relative to JAK2^V617F− cells, but this differential is abolished by TNFR2 inhibition. a BM cells from mice with JAK2^V617F-induced MPN was cultured for 16 h  ±TNFR BAs (10 μg/mL). Cells were then sorted for Lin⁻Kit⁺ expression, then subdivided based on GFP, resulting in six groups. Three independent experiments were performed. RNA was extracted for all 18 samples and subjected to microarray analysis using Affymetrix mouse 430 2.0 arrays. One sample, Untreated-JAK2^V617F+ replicate #3 failed quality standards and was removed from further analysis. b Unsupervised clustering of the remaining 17 samples grouped all samples according to genotype (JAK2^V617F) and treatment. c Fold change (FC) and P-values were generated for each condition relative to the JAK2^V617F− (Untreated) group. Sequential filters were applied to identify genes whose expression is dysregulated in JAK2^V617F+ cells and restored with TNFR BA treatment. The set was limited to genes that had a P-value of < 0.05 when compared between any two treatment groups. Then genes were limited to those that had a FC > |1| in JAK2^V617F+ relative to JAK2^V617F− cells. Genes were then subdivided into those whose expression was reversed with either TNFR1 or TNFR2 BA in JAK2^V617F+ toward JAK2^V617F− by ≥75%. To further identify those that were selectively regulated in JAK2^V617F+ cells, those with a FC > |1| in JAK2^V617F− cells were eliminated. For each of these gene sets we ranked the top 10 up- or downregulated genes. Xiap and Mapk8 were the two top differentially expressed genes between JAK2^V617F+ and JAK2^V617F− whose expression was normalized with TNFR2 BA treatment but not TNFR1 BA treatment.