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. 2016 Sep 1;7:12702. doi: 10.1038/ncomms12702

Figure 6. ORP4L is essential for T-ALL cell survival.

Figure 6

(a) Proliferation assay in Jurkat T-cells with ORP4L knockdown (left) or overexpression (right). (b) Cell death analysis in Jurkat T-cells with ORP4L knockdown for 72 and 120 h. (c) Cell death analysis in primary T-ALL cells from 10 specimens with ORP4L knockdown for 120 h. (d) The proportion of mice engrafted with primary T-ALL cells (left), and engrafted primary T-ALL cells as a percentage of the total white blood cells (right), (n=9 of shNT mice, n=3 of shORP4L mice). (e) Flow cytometric analysis of engrafted primary T-ALL cells in the blood of NOD/SCID mice with representative dot plots are shown. Western blot indicates ORP4L knockdown efficiency of T-ALL cells after infected with lentivirus for 72 h. (f) Western blot analysis of p-AMPK, LC3 and p-mTOR in control and ORP4L knockdown Jurkat T-cells (left). Confocal images of LC3 puncta in control and ORP4L knockdown Jurkat T-cells (middle and right). Arrows show LC3 puncta (200 cells from three experiments with 10 random fields/experiment). Scale bars, 10 μm. (g) Western blot of p-AMPK and LC3 in control and ORP4L knockdown Jurkat T-cells with or without AMPK inhibitor compound C (comp C, 15 μM, 4 h). (h) Cell death analysis in Jurkat T-cells with or without autophagy inhibitor, 3-MA. After infection with shNT or shORP4L lentivirus for 48 h, cells were incubated with or without 5 mM 3-MA for further 72 h, and then used for cell death analysis. The Western blot at the bottom verifies that 3-MA inhibited autophagy formation. (i) Western blot of p-PDH, p-AMPK and LC3 in primary T-ALL cells with ORP4L knockdown. The data represent mean±s.d. value from an experiment performed in triplicate. *P<0.05, **P<0.01, ***P<0.001, Student's t test.