Fig. 4: RNA sequencing revealed that CXCR3 plays an important role in the synergistic effects of AC220 and DAPT via reduced ERK activity.

MOLM13 and MV4-11 cells were treated with AC220 (2.5 nM) either alone or in combination with DAPT (2.5 µM) for 12 h, and total RNA was extracted from cultured cells for RNA-seq. a Schematic view of the study design. b Principal component analysis showed that AC220 treatment-induced major changes in the gene expression pattern in both MOLM13 and MV4-11 cell lines. Points represent each sample. The samples in one group are indicated by the same color. c The top 20 most enriched pathways identified by KEGG pathway analysis of MOLM13 DMSO-versus-Combo DEGs or MV4-11 DMSO-versus-Combo DEGs. The Q-value is indicated by the scale bar, and the size of the dots reflects the gene number. d Genes that shared the same expression pattern of AC220-versus-Combo in both MOLM13 and MV4-11 cell lines were selected (left), and the expression patterns are listed on the right. e The plot shows the GSEA of leukocyte chemotaxis genes that were differentially expressed. Data obtained from both MOLM13 and MV4-11 cells were amalgamated for GSEA. The normalized enrichment score (NES) and statistical significance/false-discovery rate Q-value (FDR) are indicated. f The publicly available dataset GSE29544 was analyzed to assess the effect of GSI treatment on CXCR3 expression. CUTLL1 cells were treated with vehicle or GSI for 3 days, and the expression of CXCR3 is presented (n = 3). Student’s t test was used for comparisons between two groups. (***P < 0.001). g GSE61715 was analyzed to assess the potential role of CXCR3 in FLT3-TKI resistance. The expression of CXCR3 in MV4-11 cells that were either sensitive or resistant to PKC412 is shown (n = 2). The NCBI GEO2R online tool was used to analyze the dataset. (*P < 0.05). h MOLM13 and MV4-11 cells were treated with AC220 (2.5 nM) and/or DAPT (2.5 µM) for 12 h, and the mRNA expression of CXCR3 was measured in triplicate by qPCR relative to GAPDH expression. Error bars indicate the average fold change vs. DMSO control ±SD. (***P < 0.001). i Expression of CXCR3 and HES1 in GFP + MOLM13 cells expressing pCDH or pCDH-DNMAML was determined by western blotting. j Expression of CXCR3, phospho-ERK (pERK), total ERK, and GAPDH in MOLM13 and MV4-11 cells was determined by western blotting following the same treatment as described. Images are representative of three independent experiments.