Fig. 2

HOTAIRM1 expression is induced by mutant NPM1 via KLF5-dependent transcriptional regulation. a-b HOTAIRM1 expression was measured by qRT-PCR in OCI-AML3 cells transfected with shNPM1 (a) and in OCI-AML2 cells transfected with the NPM1-mA plasmid (b). c qRT-PCR was conducted to determine the level of HOTAIRM1 in OCI-AML3 cells treated with KPT-330. d-e Relative luciferase activity was analyzed in NPM1-mA-silenced OCI-AML3 cells transfected with the HOTAIRM1 promoter construct (d) and NPM1-mA-enforced OCI-AML2 cells transfected with the HOTAIRM1 promoter construct (e). f Luciferase reporter assays were performed with KPT-330-treated OCI-AML3 cells using the indicated reporters. g Prediction of KLF5 binding sites in the HOTAIRM1 promoter region using JASPAR. h Enrichment of KLF5 on the E2 fragment of the HOTAIRM1 promoter was measured by ChIP in OCI-AML3 cells. i Construction of the luciferase reporter vectors HOTAIRM1-pGL3-F (containing all KLF5 binding sites), HOTAIRM1-pGL3-S1 (containing the sites at bp − 269 and − 278) and HOTAIRM1-pGL3-S2 (containing the sites at bp − 541 and − 550). j Luciferase reporter assays of OCI-AML3 cells transfected with the HOTAIRM1-pGL3-F, HOTAIRM1-pGL3-S1, or HOTAIRM1-pGL3-S2 vector, the KLF5 construct, or empty vector. k Enrichment of KLF5 on the HOTAIRM1 promoter in OCI-AML3 cells treated with or without shNPM1 was detected by ChIP. l HOTAIRM1 promoter activity in OCI-AML3 cells treated with or without shNPM1 was detected by luciferase reporter assays. m Western blot (left) and qRT-PCR (right) analyses of KLF5 expression in OCI-AML3 cells transduced with control shRNA or shNPM1. n KLF5 protein expression in transfected OCI-AML3 cells after treatment with MG132. o KLF5 expression in transfected OCI-AML3 cells treated with CHX. p-q IP assays were used to determine the binding of NPM1-mA to WWP1 (p) and the binding of KLF5 to WWP1 (q) in OCI-AML3 cells. r The binding of WWP1 to KLF5 in NPM1-silenced OCI-AML3 cells was investigated by IP assays. s Western blot analysis of endogenous KLF5 ubiquitination in transfected OCI-AML3 cells followed by siWWP1 and/or shNPM1. The data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001. n.s. indicates no significant difference