Fig 2: Activation of Akt1, Akt2, and Akt3 in a murine model of acute kidney injury (AKI)-to-chronic kidney disease (CKD) progression. (A) Representative images of Western blots for phosphorylated Akt1 (p-Akt1). Six weeks after unilateral ischemia-reperfusion injury (UIRI), the expression of p-Akt1 increased in wild-type (WT) UIRI mice compared to that in WT sham mice. (B) Representative images of immunohistochemistry for p-Akt1 (magnification ×200). The levels of p-Akt1 were higher in the renal tubules of WT UIRI mice than those in the renal tubules of WT sham mice. (C) Representative images of Western blots for p-Akt2. The levels of p-Akt2 were similar among the four groups. (D) Representative images of Western blots for Akt3. No Akt3 expression was detected in all four groups. Data are expressed as mean ± SD (n = 6/group). ap < 0.05 as compared with WT and Akt1−/− sham mice, bp < 0.05 as compared with WT UIRI mice.

Fig 3: Analysis of Akt1, Akt2, AMPKα1, and AMPKα2 expression in TNBC lymph node and lung metastases. (A) IHC scoring of Akt1, Akt2, AMPKα1, and AMPKα2 cytoplasmic expression in TNBC lymph node metastases. Samples were scored on scales of 0-3 for both intensity and distribution percentage, and the values were added together (n = 45). (B) Subcellular localization of Akt1, Akt2, AMPKα1, and AMPKα2 in TNBC lymph node metastases (n = 45). (C) IHC scoring of Akt1 and Akt2 cytoplasmic expression in TNBC lung metastases. Samples were scored on scales of 0-3 for both intensity and distribution percentage, and the values were added together (n = 6). (D) Subcellular localization of Akt1 and Akt2 in TNBC lung metastases (n = 6). IHC, Immunohistochemistry; TNBC, triple negative breast cancer.

Fig 4: Phosphorylation profiles of Akt of HeLa cells with the enforced expression of PIK3CA mutants, which were stimulated with 100 µM EGF for 30 min. (A) Expression levels of Halo-tagged PIK3CA, Akt (pan), p-Akt (Ser473), Akt1, p-Akt1, Akt2, and p-Akt2 were determined by western blotting. α-tubulin was used as a loading control. (B) Graphs depict densitometric analysis of the western blots. The intensities of the p-Akt, p-Akt1, and p-Akt2 bands were quantified by densitometry and normalized to those of α-tubulin. PIK3CA, p110α phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Fig 5: Anti-proliferative activity of alpelisib in canine HSA cell lines. PIK3CA wild-type or heterozygous/homozygous mutant cell lines were treated with increasing concentrations of alpelisib (0, 1, 2, 5, 10, 20, 50 and 100 µM) for (A) 24, (B) 48 and (C) 72 h. Cells were also treated with doxorubicin (0, 0.1, 0.5, 1, 5, 10 and 50 µM) for 24 h (D). The IC50 values and cell viability using an MTT assay were determined by measuring the absorbance at 560 nm on a microplate reader. The values of no treatment were 100%, and the values are shown from four independent experiments as the mean ± SD. (E) Western blot analysis of Akt phosphorylation in canine HSA cell lines with 10 µM alpelisib treatment for 6 h. (F) Graphs depicting densitometric analysis of the western blots compared with the loading controls. The intensities of the p-Akt, p-Akt1, and p-Akt2 bands were quantified through densitometry and normalized to the intensity of α-tubulin. PIK3CA, p110α phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; HSA, hemangiosarcoma.