Fig. 6: Gene expression profile analysis of FTY720 treated KMT2A-R leukemic cells.

A Volcano plot of gene expression differences for THP1 cells treated with FTY720 for 24 h. The dots to the left of 0 represent gene probes with adjusted p value for multiple testings (padj) < 0.05 and fold change >1.3 (log2fold change < 0). The dots to the right of 0 represent gene probes with padj < 0.05 and fold change >1.3 (log2fold change > 0). B Immunoblot for phospho-pLK1 (Thr210) (58 kDa), PLK1 (58 kDa) and GAPDH (37 kDa) in K562, Kasumi1, THP1 and MV411 upon 5 µM FTY720 treatment for 24 h. Densitometry analysis was conducted by LI-COR Image Studio software. GAPDH was used as a loading control. C qRT-PCR showing the expression of SET, MYC, HOXA9, and HOXA10 in leukemic cells upon 5 µM FTY720 treatment for 24 h. Gene expression was normalized by GAPDH control and analyzed by Pfaffl equation. Values are expressed relative to vehicle controls. Data represent mean ± SD of three independent experiments. Two tailed paired t test *p < 0.05; **p < 0.01; ***p < 0.001. D Immunoblot for SET and MYC in leukemic cells upon FTY720 5 µM treatment for 48 h. Densitometry analysis was conducted by LI-COR Image Studio software. GAPDH was used as a loading control. Values are expressed relative to vehicle control. E, F qRT-PCR showing the expression of SET, MYC, HOXA9, and HOXA10 in leukemic cells transduced with lentiviral vectors expressing shSET and selected with puromycin for 72 h. Gene expression was normalized by GAPDH control and analyzed by Pfaffl equation. Values are expressed relative to shSCRAMBLE controls. Data represent mean ± SD of two independent experiments. Two tailed paired t-test ***p < 0.001; ****p < 0.0001. G Chromatin immunoprecipitation (ChIP) experiments were performed by using anti-MLL (KMT2A/MLL) and anti-SET (SET) antibodies (Ab); ChIP with anti-IgG (IgG) represents the negative control. ChIP data are expressed as percentage of specific target gene promoter elements (pr) (i.e., HOXA9-pr, HOXA10-prE1, HOXA10-prE2, HOXA10-prE3, ACTB-pr) in precipitated chromatin compared with the INPUT (% INPUT), where ACTB-pr (the promoter of ACTIN) was the negative control. Results represent the mean ± SD average of three independent experiments. H–K Co-immunoprecipitation experiments were performed using whole cell extracts (WCE), anti-MLL or anti-SET antibodies (Ab). MLL, MLL-AF9 and MLL-AF4 proteins were immunoprecipitated as baits with the anti-MLL Ab (IP MLL) in the respective cell lines and the presence of SET was revealed by western blot (WB) (left panels); SET was immunoprecipitated as bait (IP SET) with the anti-SET Ab and the presence of wild type MLL or MLL-AF9 or MLL-AF4 was analyzed by WB with the anti-MLL Ab (right panels). Immunoprecipitation with anti-IgG (IP IgG) was used as negative control.