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. 2020 Nov 5;11(11):951. doi: 10.1038/s41419-020-03146-w

Fig. 3. GDC-0349 blocks Akt-mTOR activation in NSCLC cells.

Fig. 3

A549 cells (a) or the primary human NSCLC cells (NSCLC-1/-2) (b and c) were treated with GDC-0349 (“GDC”, 25/100 nM) for 2 h, expression of listed proteins was tested by Western blotting assays. A549 cells (d–f) or NSCLC-1 cells (g–i) were treated with 100 nM of GDC-0349 (“GDC”), rapamycin (“RAP”), perifosine (“Prf”) or AZD-2014 (“AZD”), cells were further cultured for 48–72 h, cell viability, death and apoptosis were tested by CCK-8 (d and g), Trypan blue staining (e and h) and nuclear TUNEL staining (f and i) assays, respectively. Stable A549 cells bearing a constitutively-active Akt1 (ca-Akt1, S473D) or empty vector (“Vector”) were treated with or without GDC-0349 (“GDC”, 100 nM), cells were further cultured for applied time periods, expression of listed proteins was shown (j); Cell death (Trypan blue ratio, k) and apoptosis (nuclear TUNEL ratio, l) were tested. Stable A549 cells with the CRISPR/Cas9-Akt1-KO-GFP construct (ko-Akt1 cells) were treated with or without GDC-0349 (“GDC”, 100 nM), control cells were transduced with CRISPR/Cas9 control construct (Cas9-C); Cells were further cultured for applied time periods, expression of listed proteins was shown (m); Cell death (Trypan blue ratio, n) and apoptosis (nuclear TUNEL ratio, o) were tested. The data are presented as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “Ctrl” cells (d–i). #p < 0.05 vs. GDC-0349 only treatment (d–i). *p < 0.05 vs. “Ctrl” treatment in “Vector” cells (k and l). #p < 0.05 vs. GDC-0349 treatment in “Vector” cells (k and l). The experiments were repeated five times with similar results obtained.