Figure 5
From: FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2
FilGAP partial PH-deleted isoform activates mTORC1/2. (A) Schematic diagram of FilGAP isoforms expressed in KINGS-1 cells. (B) RT-PCR analysis of expression of FilGAP isoforms in A7 melanoma, MDA-MB-231 breast cancer, U87MG glioma and KINGS-1 glioma cell line. β-actin was used as a loading control. (C) Quantification of band intensity in (B). (D) KINGS-1 cells were transfected with siRNA targeted against all FilGAP isoforms (FilGAP KD#1) or only isoform 1. (E) P-S6K/Total S6K and P-AKT/Total AKT ratio in (D) were calculated (n = 3). (F) FilGAP isoform expressions in (D) were calculated. (G) Box and jitter plots show expression level of overall ARHGAP24, isoform1, isoform3, and isoform4 in grade II (Astrocytoma), III (Anaplastic Astrocytoma), and IV (Glioblastoma multiforme). The data was obtained from TCGA database. Significance was determined using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. (H) KINGS-1 cells were transfected with HA-FilGAP rKD#1 constructs (WT, 96-748aa) after depletion of FilGAP with siRNA. (I) P-S6K/Total S6K and P-AKT/Total AKT ratio in (H) were calculated (n = 8). (J) HEK293T cells were transfected with HA-FilGAP (isoform 3: 96-748aa or ΔPH:131-748 aa) and Flag-Rictor. 24 h after transfection, cells were lysed and after immunoprecipitation with anti-FilGAP antibody. *P < 0.05, **P < 0.01 , ***P < 0.001, ****P < 0.0001(Student’s t-test, E, F, I).
