Fig 1: Rho GTPase activating protein 25 (ARHGAP25) inhibited AKT/mTOR signaling pathway. A, Gene set enrichment analysis (upper panel) showed the enrichment of AKT/mTOR signaling in PAAD tumors with downregulated ARHGAP25 expression. Differential expressions (lower panel) of the gene signature (CREIGHTON_AKT1_SIGNALING_VIA_MTOR_DN) are shown in the heatmap created by the GSEA software. B,C, Protein levels of ARHGAP25, total and phosphorylated AKT, and mTOR were examined by western blotting in PAAD cells. In sh-ARHGAP25 BxPC-3 cells, the expressions of p-AKT and p-mTOR were significantly increased (B). In ARHGAP25-overexpresisng AsPC-1 cells, the expressions of p-AKT and p-mTOR were significantly decreased (C). D,E, Western blotting was used to determine protein expression in xenografts. The expressions of p-AKT and p-mTOR were significantly decreased in the ARHGAP25-OE xenografts. (n=6) (D). Conversely, the expressions of p-AKT and p-mTOR were significantly increased in the xenografts with ARHGAP25 knockdown (n=6) (E). Data are shown as the mean ± SD. **P < .01, ***P < .001.
Fig 2: Forest plot of the multivariate analysis data of the prognostic value of ARHGAP25 expression for predicting overall survival in patients with colorectal cancer. M0, no variables; M1, age and sex; M2, invasion degree, lymph node metastasis, distant metastasis and TNM stage; M3, tumor location, histological type, histological grade and tumor deposits; M4, chemotherapy and TCM treatment; M5, age, sex, invasion degree, lymph node metastasis, distant metastasis, TNM stage, tumor location, histological type, histological grade, tumor deposits, chemotherapy and TCM treatment. ARHGAP25, Rho GTPase-activating protein 25; TCM, traditional Chinese medicine.
Fig 3: Rho GTPase activating protein 25 (ARHGAP25) suppressed glycolysis in PAAD in vitro. A, Protein levels of ARHGAP25, HIF-1α, PKM2, and LDHA were examined by western blotting in PAAD cells. In ARHGAP25-overexpresisng AsPC-1 cells, the expressions of HIF-1α, PKM2, and LDHA were significantly decreased. B, Glucose uptake was measured using the 2-NBDG uptake assay kit by flow cytometry. Overexpression of ARHGAP25 significantly decreased 2-NBDG uptake (n=3). C,D, Extracellular lactate levels and intracellular ATP levels were measured using the lactate assay kit and the ATP assay kit, respectively. Lactate production (C) and intracellular ATP levels (D) were significantly decreased in ARHGAP25-overexpresisng AsPC-1 cells (n=3 per group). E, In sh-ARHGAP25 BxPC-3 cells, the expressions of HIF-1α, PKM2, and LDHA were significantly increased. F,G, Lactate production (F) and intracellular ATP levels (G) were significantly increased in sh-ARHGAP25 BxPC-3 cells (n=3 per group). H, ARHGAP25 knockdown significantly increased 2-NBDG uptake (n=3). Data are shown as the mean ± SD. **P < .01, ***P < .001.
Fig 4: Rho GTPase activating protein 25 (ARHGAP25) suppressed glycolysis in PAAD in vivo. A,B, Western blotting was used to determine protein expression in xenografts. The expressions of HIF-1α, PKM2, and LDHA were significantly decreased in the ARHGAP25-OE xenografts (n=6). C,D, The expressions of HIF-1α, PKM2, and LDHA were significantly increased in the xenografts with ARHGAP25 knockdown (n=6). Data are shown as the mean ± SD. *P < .05, ***P < .001.
Fig 5: Rho GTPase activating protein 25 (ARHGAP25) attenuated cytotoxic effects of gemcitabine or 5-fluorouracil in pancreatic adenocarcinoma (PAAD) cells. A,B, ARHGAP25-overexpressing AsPC-1 cells were treated with gemcitabine (0.5µM) (A) and 5-fluorouracil (5 µM) (B) for 72 h. Upregulation of ARHGAP25 enhanced both gemcitabine and 5-fluorouracil cytotoxicity in AsPC-1 cells. C,D, ARHGAP25-knockdown BxPC-3 cells were treated with gemcitabine (5 µM) (C) and 5-fluorouracil (10 µM) (D) for 72 h. ARHGAP25 knockdown reduced the chemosensitizing effect of gemcitabine or 5-fluorouracil. The cell viability was measured by CCK-8 assay at 450nm. Data are shown as the mean ± SD. **P < .01, ***P < .001.
Supplier Page from Abcam for Anti-ARHGAP25 antibody [EPR13233-60]