Fig 1: GSE1 downregulation contributes to the myeloid differentiation reactivation enforced by LSD1 inhibition.A Percentage of NB4 CD11b-positive cells measured by flow cytometry 48 h post transduction with the shA1 and shB2 cloned in pLKO.1 puro, or with the empty pLKO.1 puro vector (EV) used as negative control. Dot plots show the results obtained in one of the two biological replicates of the experiment. The bar chart represents mean ± SD (n = 2 biological replicates). B Left panel: Percentage of CD11b- and CD14- positive THP-1 cells, assessed by flow cytometry analysis 48 h upon transduction with pLKO.1 puro vector containing shA1, shB2 or EV as negative control. Dot plots display the data of one of the two biological replicates of the experiment. The bar chart represents the mean + SD from two (n = 2) biological replicates. Right panel: Bar graph of the fold change (FC) of CD14 mean fluorescence intensity in THP-1 cells transduced for 48 h with shA1 and shB2, over the corresponding level in the EV-transduced cells. The chart represents mean + SD (n = 2 biological replicates). C Left panel: Representative dot plots showing the levels of CD11b- and CD86- positive cells as well as the intensity of these markers 48 h and 72 h post transduction with the shA1 and shB2 constructs targeting GSE1, or a nonspecific shRNA construct (NTC) cloned in pLKO.1 puro vector as negative control in the BB104 MLL- translocated primary patient sample. Right panel: Bar graph displaying the mean fluorescence intensity of CD11b, CD86 and CD14 in BB104 primary patient cells transduced for 48 h and 72 h with NTC, shA1 and shB2. The results are plotted as FC of the mean fluorescence intensity of these markers in the GSE1 KD samples over the corresponding levels in the NTC-transduced cells. The chart represents mean ± SD (n = 2 biological replicates). D Percentage of BB104 CD14-positive cells measured by flow cytometry at 48- and 72-h post transduction with the shA1 and shB2 cloned in pLKO.1 puro, or with the NTC used as negative control. Data are plotted as FC of the CD14 mean fluorescence intensity in the GSE1 KD samples over the corresponding levels in the NTC-infected cells. The chart represents mean ± SD (n = 2 biological replicates). E Percentage of CD11b-positive cells evaluated by flow cytometry in control (EV) and NB4 GSE1 OE cells, treated for 24 h with MC2580 (2 µM). NB4 GSE1 OE is produced by transducing the pLEX_307 vector with the GSE1 coding sequence (NM_001134473.3), while control cells are transduced with the pLEX_307 EV. The chart represents mean ± SD (n = 4 biological replicates; paired T-test, **p-value < 0.01). F Left panel: Flow cytometry analysis of the percentage of CD11b- and CD14- positive cells in EV and GSE1 OE THP-1 cells treated for 24 hours with MC2580. THP-1 GSE1 OE is generated by lentiviral transduction of the pLEX_307 vector including GSE1 coding sequence (NM_001134473.3), while control cells are transduced with the pLEX_307 EV. The chart represents mean ± SD (n = 3 biological replicates; paired T-test, *p-value < 0.05). Right panel: Bar graph displaying the CD14 mean fluorescence intensity in DMSO, MC2580 and MC2580 + GSE1 OE THP-1 cells. The chart represents mean ± SD from three (n = 3) biological replicates (paired T-test, *p-value < 0.05).