Fig 2: Gastric cancer cells lacking E-cadherin have enhanced sensitivity to the allosteric AKT inhibitors ARQ092 and MK2206. (A) Western blots of pAKT-Ser473, pan-AKT, and ß-actin in NCI-N87-WT and CDH1-/- cells. (B) Relative expression of pAKT and pan-AKT in NCI-N87-WT and CDH1-/- cells. (C–F) Normalised NCI-N87-WT and CDH1-/- cell counts 48 h after treatment with serial dilutions of ARQ-092 (C), MK2206 (D), perifosine (E), and SC66 (F). Wild-type, black bars; CDH1-/-, grey bars. Six fields per well at 4× magnification were captured using the Cytation 5 imager (Biotek). Nuclei were counted using Gen5 (Biotek, Winooski, Vermont) and normalised to the vehicle control for each cell line. (For all graphs, error bars = SEM; ns = p > 0.05, * = p < 0.05, ** = p < 0.01, n = 3 independent biological replicates; unpaired two-sided t-test.)

Fig 3: Non-malignant breast cells lacking E-cadherin exhibit a higher sensitivity to the allosteric AKT inhibitors ARQ-092, MK2206, perifosine, and SC66. (A–F) Normalised MCF10A-WT and CDH1-/- cell counts 48 h after treatment with serial dilutions of Ipatasertib (A), ARQ-092 (B), MK2206 (C), Perifosine (D), SC66 (E), and PHT-427 (F). Wild-type, black bars; CDH1-/-, grey bars. Six fields per well at 4× magnification were captured using the Cytation 5 imager (Biotek). Nuclei were counted using Gen5 (Biotek) and normalised to the vehicle control for each cell line. (G) Western blots of pAKT-Ser473 and pan-AKT levels in untreated MCF10A-WT and CDH1-/- cells. (H) Relative expression of pAKT and pan-AKT in MCF10A-WT and CDH1-/- cells. (I) Western blots of pAKT-Ser473 and pan-AKT levels in MCF10A-WT and CDH1-/- cells after 4 h of DMSO or allosteric AKT inhibitor treatments; vehicle (0.1% DMSO), ARQ092 (2 µM), MK2206 (6.25 µM), perifosine (7.5 µM), and SC66 (0.78 µM). (For all graphs, error bars = SEM; ns = p > 0.05, * = p < 0.05, ** = p < 0.01, n = 3 independent biological replicates; unpaired two-sided t-test.)

Fig 4: CD28 costimulation increases glycolysis and glucose transporters.(A) Glycolytic stress test results showing representative ECAR and OCR (±SEM) normalized to cell count using Cytation 5 (BioTek). CD8+ RCC TIL glycolysis following IL-7 (gray), CD3 alone (black), CD3 with CD28 costimulation (red). n = 5. *P < 0.05, ***P < 0.001 by 1-way ANOVA with Tukey’s post hoc test. (B) Flow cytometry analysis showing MFI of GLUT1 and GLUT3 normalized to IL-7 and comparing CD3 with CD28 costimulation (red). n = 9. **P < 0.01 by 1-way ANOVA with Tukey’s post hoc test.

Fig 5: Discovery of a novel small-molecule antiviral. (A) Structure of compound 1 (F2205-0189), compound 3 (3G), ERDRP-0519 (16), and JMN3-003 (19). (B) Assessment of the compounds’ inhibitory impact on virus-induced syncytium formation. Microscopic images of cells infected with OPneon in the presence of the compounds. Scale bars, 2,000 µm. Pictures were captured with a Cytation 5 imaging multimode reader (BioTek). Note that due to automatic settings, discrepancy in background intensity measurement is visible in the stitched images. (C) IC50 measurement of compounds against the attenuated OP-CDV strain. (D) IC50 measurement of compounds against the wild-type A75/17-CDV. (E) Measurement of the cytotoxic effect of the inhibitors. Ninety-five percent confidence intervals are shown in parentheses. Relative luminescence values were normalized for values obtained in the presence of DMSO control and represent means from three independent experiments. (F) Impact of compound 1 on cell cycle progression of treated cells. Vero cells were incubated in the absence or presence of increasing concentration of compound 1 for 42 h and analyzed using flow cytometry. The values show the means ± SD from three independent experiments. Dunnett’s multiple-comparison test was applied after two-way analysis of variance (ANOVA) (*, P < 0.05). (G) Assessment of viral proliferation after incubation with the compound. Vero cells expressing canine SLAM (cSLAM) were infected with wild-type A75/17-CDV (A75neon/nLucP) at an MOI of 0.01 in the presence of increasing concentrations of the compound. After 30 h, infected cells were frozen and thawed (twice), and viruses in the lysates were harvested and titrated in Vero-cSLAM cells.