Figure 4.
ORP4L interacts with PI3Kδ to enhance PI(3,4,5)P3 generation and AKT activation in malignant transformed T cells. (A) Coimmunoprecipitation (top) and confocal microscopy (bottom) analysis of ORP4L binding to and colocalization with PI3Kδ in MT-4 cells. Scale bar, 10 μm. (B) The binding model of ORP4L (yellow) to PI3Kδ (blue) computationally predicted. The key interaction residues of ORP4L (yellow) and PI3Kδ (blue) complex is shown. (C) The root-mean-square deviation values of PI(4,5)P2 in PI3Kδ and in the ORP4L-PI3Kδ complex. (D) The binding model of ORP4L (yellow) to PI(4,5)P2 (red)/PI3Kδ (blue) complex computationally predicted. The site of PI(4,5)P2/PI3Kδ/ORP4L complex is shown. (E-F) PI(4,5)P2 (E) and PI(3,4,5)P3 (F) contents in T cells isolated from sick B-NDG mice in Figure 3. Scale bars, 10 μm. The panels on the right indicate quantitation of relative fluorescence intensity. Mean ± standard deviation (SD; n = 15-20 cells; Student t test). (G) Phosphorylated AKT, p65, and p53 levels in T cells isolated from the sick B-NDG mice in Figure 3. (H-I) PI(4,5)P2 and PI(3,4,5)P3 contents in T cells of WT, LCK/R26Tax, and ORP4Lcko;LCK/R26Tax mice at the age of 15 months. Representative images (H) and the relative quantification of fluorescence intensity (I) are shown. Scale bars, 10 μm. Mean ± SD (n = 10 cells; Student t test). (J) AKT activation and p65 and p53 phosphorylation in T cells of WT, LCK/R26Tax, and ORP4Lcko;LCK/R26Tax 15-month-old mice. (K) PI(4,5)P2 and PI(3,4,5)P3 contents in human normal T cells or the T cells of a patient with ATL (ATL#13) transduced with lentivirus-carrying control (shNT) or ORP4L-specific short hairpin RNA (shORP4L). The T cells were transduced and cultured in vitro for 72 hours before analysis. Mean ± SD (n = 10 cells; Student t test). (L) AKT activation, p65 and p53 phosphorylation in T cells treated as in panel K. ***P < .001.

ORP4L interacts with PI3Kδ to enhance PI(3,4,5)P3 generation and AKT activation in malignant transformed T cells. (A) Coimmunoprecipitation (top) and confocal microscopy (bottom) analysis of ORP4L binding to and colocalization with PI3Kδ in MT-4 cells. Scale bar, 10 μm. (B) The binding model of ORP4L (yellow) to PI3Kδ (blue) computationally predicted. The key interaction residues of ORP4L (yellow) and PI3Kδ (blue) complex is shown. (C) The root-mean-square deviation values of PI(4,5)P2 in PI3Kδ and in the ORP4L-PI3Kδ complex. (D) The binding model of ORP4L (yellow) to PI(4,5)P2 (red)/PI3Kδ (blue) complex computationally predicted. The site of PI(4,5)P2/PI3Kδ/ORP4L complex is shown. (E-F) PI(4,5)P2 (E) and PI(3,4,5)P3 (F) contents in T cells isolated from sick B-NDG mice in Figure 3. Scale bars, 10 μm. The panels on the right indicate quantitation of relative fluorescence intensity. Mean ± standard deviation (SD; n = 15-20 cells; Student t test). (G) Phosphorylated AKT, p65, and p53 levels in T cells isolated from the sick B-NDG mice in Figure 3. (H-I) PI(4,5)P2 and PI(3,4,5)P3 contents in T cells of WT, LCK/R26Tax, and ORP4Lcko;LCK/R26Tax mice at the age of 15 months. Representative images (H) and the relative quantification of fluorescence intensity (I) are shown. Scale bars, 10 μm. Mean ± SD (n = 10 cells; Student t test). (J) AKT activation and p65 and p53 phosphorylation in T cells of WT, LCK/R26Tax, and ORP4Lcko;LCK/R26Tax 15-month-old mice. (K) PI(4,5)P2 and PI(3,4,5)P3 contents in human normal T cells or the T cells of a patient with ATL (ATL#13) transduced with lentivirus-carrying control (shNT) or ORP4L-specific short hairpin RNA (shORP4L). The T cells were transduced and cultured in vitro for 72 hours before analysis. Mean ± SD (n = 10 cells; Student t test). (L) AKT activation, p65 and p53 phosphorylation in T cells treated as in panel K. ***P < .001.

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