Flow cytometry software

5637 cells transfected with MAT1A plasmid or a control empty vector were seeded at 20% confluency (n = 6) and dyed using the CellTrace™ CFSE Cell Proliferation Kit, to quantify cell proliferation. All cells were collected three days post-transfection and immediately analyzed for fluorescence using a FACSMelody™ flow cytometer (Becton, Dickinson & Co, Franklin Lakes, NJ). Expansion indices as well as proliferation/division indices were determined using cell proliferation modeling generated by FlowJo flow cytometry software (FlowJo, LLC, Ashland, OR, USA). Statistics were performed using a Student’s t-test including n = 6 biological replicates from each 5637 and 5637^MAT1A+ cells.

Morphologic differentiation of OCI-AML3, IMS-M2, K562 or patient cells was determined by CD11b expression and morphologic assessment of cells stained with May-Grünwald-Giemsa. Cells were treated for 48 h and 72 h with either 500 nM OTX015 (MK-8628), 500 nM JQ1, 1 µM ATRA + 1 µM ATO, or 0.1% DMSO followed by flow cytometric detection of the differentiation marker CD11b Alexa Fluor^® 488 (#557701, Becton Dickinson, Le Pont de Claix, France) using a CytoFLEX flow cytometer (Beckman Coulter, Villepinte, France) and analyzed with the FlowJo flow cytometry software (FlowJo LLC, Ashland, OR, USA). For morphologic analysis, cytospin preparations (Cytospin 4, Fisher Scientific, Illkirch, France) of OCI-AML3, IMS-M2, K562 or patient cells treated for 48 h by OTX015 (MK-8628) 500 nM, JQ1 500 nM, ATRA 1 µM + ATO 1 µM, or 0.1% DMSO were stained with May-Grünwald Giemsa solution and examined under light microscopy with a Zeiss microscope (Carl Zeiss, Marly le Roi, France) with a X40 objective.