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. 2019 Mar 21;216(5):1120–1134. doi: 10.1084/jem.20172170

Figure 3.

Figure 3.

PIP4K2A negatively regulates cellular proliferation, clonogenic growth, and the AKT signaling pathway in patient-derived primary GBMs. (A) Effects of PIP4K2A on in vitro proliferation of GBM cells that were transduced with either control (non-target) or PIP4K2A-expressing virus. Values are presented as mean ± SD (n = 5). (B) PIP4K2A suppresses tumorsphere formation. Bar, 100 µm. (C and D) LDA for in vitro tumor sphere formation. LDA clonogenic significance was measured by linear regression analysis. (E) A schematic representation of dual-color competition assay of GBM4 cells in vivo. A total of 100,000 cells from a 1:1 mixture of RFP-labeled PIP4K2A WT cells (red) and GFP-labeled control (NT) cells (green) was injected into the mouse brains. (F) Immunoblot analysis of PIP4K2A in patient-derived GBM4 cells transduced with NT control or PIP4K2A-WT vector. (G) FACS analysis of post–in vivo dual-color competition assay. (H) Bar graph represents the relative percentage of GFP- and RFP-positive cells from the FACS analysis. (I) Representative bar graph of the number of GFP- and RFP-positive cells that were counted from different regions that were selected randomly from the post–in vivo competition assay. Values are presented as mean ± SD (n = 4). (J) Immunofluorescence images of cryosectioned mouse brains from the in vivo dual-color competition assay. Bars, 1,000 (left) and 100 µm (right). (K) Representative confocal images of immunofluorescence staining of pAKT in GBM cells that were transduced with NT virus or PIP4K2A-expressing virus. Bar, 20 µm. (L) Representative immunoblot analysis of phosphorylated-AKT (pAKT), total AKT, phosphorylated-S6K (pS6K), total S6K, and PIP4K2A expression in patient-derived tumor cells and NPCs after they were transduced with either PIP4K2A or control. Actin was used as a loading control. (M) Densitometric analysis of the blots in L. Values are presented as mean ± SEM. Data shown in A–D, F, and K–M are representative of three independent and reproducible experiments. P values: A and M, two-tailed Student’s t test. *, P ≤ 0.05; ***, P ≤ 0.001. Endo., endogenous; Exo., exogenous.