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. 2015 Jul 16;5:12230. doi: 10.1038/srep12230

Figure 4. A3G promotes anoikis resistance through PTEN-mediated activation of the Akt pathway.

Figure 4

(A) Apoptosis-related proteins are involved in anoikis resistance. Western blot analysis of apoptosis-related proteins in stable A3G-expressing cells, A3G knockdown cells and control cells. Compared with the control cells, the anti-apoptotic proteins Bcl-2, Mcl-1 and phospho-Bcl-2 were up-regulated and cleaved caspase-3 was attenuated in stable A3G-expressing cells after detachment. In contrast, Bcl-2, Mcl-1 and phospho-Bcl-2 were down-regulated in stable A3G knockdown cells. GAPDH was used as a loading control. (B) Akt kinase is activated by A3G. Akt kinase activity was up-regulated in stable A3G-expressing BxPC3 cells. GAPDH was used as a loading control. (C) Detection of phospho-Akt. A3G-expressing cells and control cells were collected and seeded into poly-HEMA-coated 6-well plates in attachment and detachment conditions. The cells were incubated at 37 °C for 24 h. Western blot analysis of Akt in A3G-expressing cells and control cells. Phospho-Akt (Thr308) and phospho-Akt (Ser473) were up-regulated in A3G-expressing cells compared with control cells, both in attachment and detachment. GAPDH was used as a loading control. (D) Detection of phospho-Bad. Western blot analysis of Bad in stable A3G-expressing BxPC3 cells and control cells. Compared with control cells, phospho-Bad (Ser136) was up-regulated in stable A3G-expressing cells after detachment. GAPDH was used as a loading control. (E) Inhibition of Akt decreases anoikis resistance in A3G overexpressing cells. Cell viability of stable A3G-expressing BxPC3 cells after detachment was significantly higher than control (*aP < 0.05; Student’s t-test); cell viability of stable A3G-expressing BxPC3 cells in detached state was remarkably decreased by Akt inhibition (*bP < 0.05; Student’s t-test). All data are represented as the means ± SEM for triplicate experiments. (F) Detection of PTEN. Western blot analysis demonstrated that phospho-PTEN was up-regulated in stable A3G-expressing BxPC3 cells and SGC7901 cells transiently transfected with A3G-HA plasmid. (G) PTEN is a target of A3G-induced Akt activation. Stable A3G-expressing BxPC3 cells were lysed, and A3G was co-immunoprecipitated with HA-tag agarose-conjugated antibodies. Western blot detection of PTEN showed that A3G could interact with PTEN. All data are repeated for three times in the same condition.