New insights into the functional role of protein phosphatase 4 regulatory subunit PP4R3A/SMEK1 in the regulation of leukemic cell fate

Investigation into the Role of Long-Non-Coding RNA MIAT in Leukemia

“…5 × 10 6 cells were transfected and were incubated in complete IMDM medium supplemented with 20% heat inactivated FBS and 10 mg/mL gentamicin reagent for 24 h and re-plated at 4 × 10 6 for subsequent assessment of long- and short-term cell survival and apoptosis [ 60 , 61 ]. The efficiency of transfection for Jurkat cell line was 64–72% as determined by Cy3 labelling (Invitrogen, Oxford, UK; #1632) [ 14 , 63 , 64 ].…”

“…Cell viability was determined by counting trypan blue solution 0.4% w / v (Sigma-Aldrich, UK, # T8154) stained samples using a hemocytometer and light microscopy. Cell viability was also determined using a commercial Muse ® Count and Viability Kit (Luminex, TX, USA; #MCH100104) and the Muse ® Cell Analyzer (Merck Millipore, Darmstadt, Germany), following manufacturer’s protocol [ 14 , 63 , 64 , 65 ].…”

“…Apoptosis was assessed by examining nuclear morphology under fluorescence microscopy following staining with acridine orange (25 μg/mL) (Sigma-Aldrich, UK; # 235474). Cells with condensed or fragmented chromatin were considered apoptotic [ 14 , 63 , 64 ].…”

“…Cell cycle analysis was performed using the Muse cell cycle kit (Luminex, Austin, TX, USA #MCH1001060) and flow cytometry, as described previously [ 14 , 63 , 64 ]. Transfected cells were plated at a density of 4 × 10 6 cells/well in 6-well plates containing 5 mL of fresh cell culture medium.…”

“…Long-term survival was assessed by the cells ability to form colonies on soft agar [ 63 , 64 ]. An equal cell number proportion of cells was diluted in 3 mL of IMDM containing 20% heat-inactivated FBS, 10% cell-specific filtered conditioned medium, and 10% noble agar solution 0.5% w / v (Difco, Omagh, UK; #214230).…”