Fig 2: Proposed model for apoptosis suppression via R-Ras pathway by RasGRP2. RasGRP2: ras guanyl nucleotide releasing protein 2, PI3K: phosphoinositide 3-kinase, JNK: c-jun N-terminal kinase, HK-2: hexokinase-2, VDAC: voltage-dependent anion channel, CTZ: clotrimazole, PARP: poly (ADP-ribose) polymerase, Q-VD-OPH: quinoline-Val-Asp-Difluorophenoxymethylketone.

Fig 3: Inhibition of Bax translocation by RasGRP2 via R-Ras-PI3K-Akt signaling pathway. (a–d) Each effect was detected by western blotting. (a) Phosphorylation of JNK was detected using a specific antibody. Cells were incubated with or without TNF-a, anisomycin, or BAM7 for 1 h. (b) Activated Bax was isolated by immunoprecipitation (IP) using a Bax (6A7) antibody. Cells were incubated with or without BAM7 or anisomycin for 16 h. (c,d) Cytosolic and mitochondrial fractions were isolated by cell fractionation kit. (d) Cells were pre-treated with siRNAs against R-Ras or negative control siRNA and incubated with or without BAM7 for 16 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin, IP: immunoprecipitation.

Fig 4: Expression of endogenous RasGRP2 in TERT HUVECs and suppression by endogenous RasGRP2 of the decreased cell viability. (a,b) Each effect was detected using western blotting. (a) RasGRP2 was detected using a specific antibody. R: RasGRP2-stable overexpression cells. (b) Activated R-Ras was isolated using a pull-down assay with RalGDS-RBD agarose beads. Cells were treated with siRNA against RasGRP2 or a negative control siRNA. (c,d) Cell viability was determined using a Cell Counting Kit-8 assay. Cells were pre-treated with an siRNA against RasGRP2 or a negative control siRNA and incubated with or without BAM7 (c: 10 μM, d: 20 μM) for 48 h. Data are shown as the mean ± SD (n = 3), **P < 0.01 compared with each cell control, and $$P < 0.01 compared with each cell transfected with the negative control siRNA.

Fig 5: Suppression of apoptosis by promoting mitochondrial hexokinase-2 translocation via RasGRP2-R-Ras-PI3K-Akt signaling pathway. (a–c) Cells were pre-incubated with or without LY294002 (b) or CTZ (c) and incubated with or without BAM7 for 16 h. Each effect was detected by western blotting. Cytosol and mitochondrial fractions were isolated by cell fractionation kit. (d) Cells were pre-incubated with or without CTZ and incubated with or without BAM7 for 48 h. Cell viability was determined by Cell Counting Kit-8 assay. M: mock cells, R: RasGRP2-stable overexpression cells, CTZ: clotrimazole, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), **P < 0.01 compared with each cell control, ##P < 0.01 compared with each R cell without pre-treatment, and ++P < 0.01 compared with each M cell without pre-treatment.