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. 2021 Mar 8;118(11):e2010206118. doi: 10.1073/pnas.2010206118

Fig. 7.

Fig. 7.

PI3K/AKT inhibitors block AKT and STAT1 activation blunting induction of the IFN response, proliferation and migration in CAV1-deficient primary human PAECs. (A) Treatment with Wortmanin (50 nM) significantly reduced AKT activation in PAECs with a trend for this effect to be larger in CAV1-silenced cells than siCTRLs (P = 0.06; for an interaction). Wortmanin also significantly reduced STAT1 activation in CAV1-deficient PAECs with no effect on siCTRL cells. Total AKT or STAT1 were not altered by Wortmanin (P ≥ 0.10). Densitometric quantification of phosphorylated and total AKT and STAT1 is normalized to β-actin and presented relative to vehicle-treated siCTRLs (mean ± SE) with representative Western blots displayed on the left. (B) CXCL10 production was reduced by LY294002 and Wortmanin in both CAV1-silenced and siCTRL transfected PAECs. CXCL10 measured by ELISA presented as mean± SE. After siRNA transfection (48 h), PAECs were serum-starved for 24 h followed by 24 h of drug treatment or vehicle control prior to whole-cell lysate or cell supernatant collection. (C) Both GDC-0980 and MK-2206, PI3K/AKT inhibitors, dose-dependently reduced cell proliferation in CAV1-silenced PAECs. Unlike GDC-0980, which reduced cell proliferation of siCTRL PAECs at high doses (P < 0.0001 for the downward trend of the slope at doses >10 nM), MK-2206 treatment did not impact proliferation in siCTRL cells (P = 0.51 for the slope). After CAV1 knockdown (48 h), PAECs were transferred to 96-well plates in complete media for 6 h. Cells were then serum-starved for 24 h, returned to complete media, and then treated with either GDC-0980, MK-2206, or vehicle control. BrdU was added after 48 h of drug treatment and incorporation was assessed 24 h later. Data for GDC-0980 (n = 4) and MK-2206 (n = 3) is presented as the mean FC ± SE. (D) Treatment with a pan-PI3K inhibitor (GDC-0980) reduced pAKTSer473 in both CAV1-silenced PAECs and siCTRLs. After siRNA transfection (48 h), PAECs were treated with drug or vehicle control for 6 h prior to whole-cell lysate collection. Representative Western blot shown from three experiments. Replication throughout represents independent experiments each using different PAEC donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.