Figure 3.

Apoptosis and induction of CDKN1A after PIP4K2A KD is dependent on MTOR. Human THP1 AML cells were infected with a lentiviral vector targeting PIP4K2A for KD, or a non-targeting control (NTC), with puromycin as the selectable marker. Cells were treated with puromycin for 48 hours to kill untransduced cells. Control and KD cells demonstrated similar viabilities at this time point, indicating equivalent transduction rates (data not shown). (a) Bar chart shows mean±SEM percentage of apoptotic cells seven days following lentiviral infection for the indicated conditions, as determined by annexin V binding (n=6). * indicates p<0.001 using one-way ANOVA followed by Fisher’s least significant difference post hoc test. (b) & (c) Western blots show expression of the indicated proteins, and in the indicated conditions, in THP1 AML cells 72 hours following lentiviral infection. Data from two separate experiments are shown (lanes 1 & 2 for each condition). (d-g) THP1 cells were double infected with lentiviral vectors targeting PIP4K2A or CDKN1A for KD, or a non-targeting control, with GFP and puromycin as selectable markers. Puromycin was added 24 hours later and viable GFP⁺ cells were FACS purified 48 hours following spinfection. Bar charts show (d) mean±SEM expression of PIP4K2A and CDKN1A relative to control cells, as determined by quantitative PCR, in FACS purified cells 72 hours following lentiviral infection (n=6); (e) mean±SEM fold change in resorufin (alamarBlue) signal of puromycin-resistant, GFP+ cells cultured for 72 hours following FACS purification (n=3); and (f) mean±SEM colony forming cell (CFC) frequencies (n=3) for the indicated control and KD conditions (n=3). For (e) and (f), * indicates p<0.01 for the indicated comparisons, as determined by an unpaired t-test. (g) Representative images from (f).