Fig. 5

SMARCA5 genetic and pharmacologic targeting reduce self-renewal of NUP98-NSD1/FLT3-ITD immortalized hematopoietic stem and progenitor cells and downregulate HoxA9 and Meis1 expression. A Western-blot analysis showing the expression of SMARCA5 in NUP98-NSD1/FLT3-ITD hematopoietic cells. B RT-qPCR analysis showing the knockdown of BPTF at mRNA level, upon 1st and 2nd methylcellulose (re)plating using 2 different primers for 2 different shRNAs. C Colony numbers after (re)plating in methylcellulose upon treatment with 2 μg/ml doxycycline. N = 3 biological replicates. T-test, ****P < 0.0005. Data are mean ± SD. D Colony formation capacity in methylcellulose after plating in presence of 2 μg/ml doxycycline. The images represent the effect observed in 3 biological replicates. E Immunofluorescence staining of NUP98-NSD1/FLT3-ITD immortalized hematopoietic cells upon doxycycline induced knockdown of SMARCA5, showing formation of the fusion’s nuclear condensates irrespectively from SMARCA5 expression. The cells were stained using Anti-FLAG and Anti-SMARCA5 antibodies, labeled with Alexa Fluor 568 secondary antibody (red), and Alexa Fluor 647 secondary antibodies (green), while nuclei were counterstained with DAPI (blue). F Proximity ligation assay in NUP98-NSD1/FLT3-ITD immortalized hematopoietic cells showing the lack of the signal for the interaction between NUP98-NSD1 and SMARCA5 in shRNA1 + DOX (SMARCA5 knockdown) condition and upon treatment with 10 μM ED2-AD101. G HoxA9 and Meis1 expression measured by RT-qPCR in cells upon induced shRNA-mediated knockdown of SMARCA5 (1 μg/ml doxycycline) and treatment with 10 μM ED2-AD101. N = 3 biological replicates. T-test, *P < 0.05. Data are mean ± SD