Fig 2: PI3Kα is required for EGF-stimulated PKB phosphorylation in MCF10a cells.MCF10a cells were serum-starved, pre-incubated with inhibitors or vehicle for 20 mins and stimulated with EGF (at the indicated doses) or its vehicle (the vehicle of the inhibitors was only was added to those samples stimulated with EGF without inhibitors or “starved”). After 15 mins the cells were lysed, aliquots were immuno-blotted with anti-β-COP (loading control, 110 kD), anti-phospho-T308-PKB and -S473-PKB antibodies simultaneously on the same filters. The immobilized antibodies were quantified with fluorescent 2° antibodies (goat-anti mouse-IRDye 800 for T308 and β-COP and goat-anti rabbit-IRDye 680 for S473 and a Li-Cor image analysis platform. Data are presented normalized to β-COP expression in the same sample. Panel A. shows a representative immuno-blot used to derive data shown in C and D. The final concentrations of the inhibitors with the cells were; A66, 6 µM; TGX221, 40 nM; IC87114, 1 µM; “mix”: A66, 6 µM+TGX221, 40 nM+IC87114, 1 µM; PI103, 1 µM. Panel B. The conditions of the experiments and the phosphorylation of S473-PKB was quantified, as in A (except, the experiment included PIK75 at 1 µM and the concentrations of A66, in µM, shown). The data are means ± SE (n = 3 experiments). The data indicate an IC50 of 800 nM. Panel C. The conditions of the experiments and the phosphorylation of S473-PKB was quantified, as in A. The data shown are means ± SE (n = 3 experiments). Panel D. The conditions of the experiments and the phosphorylation of T308-PKB was quantified, as in A. The data shown are means ± SE (n = 3 experiments).

Fig 3: Autophagy caused by MTOR inhibition does not require MAPK8/9 in immortalized MEFs. (a) The amount of RPS6KB1, p-Thr389 RPS6KB1, and TUBA (tubulin alpha) in WT and mapk8−/- mapk9−/- immortalized MEFs after incubation without or with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8−/- mapk9−/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation of the cells with 250 nM torin 1 (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression by WT and mapk8−/- mapk9−/- immortalized MEFs after incubation (2 h) without or with 250 nM torin 1 in the absence or presence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control condition (first lane). The ‘Change in MAP1LC3B-II’ was calculated by subtracting MAP1LC3B-II:GAPDH (media+ CQ condition) from MAP1LC3B-II:GAPDH (torin 1+ CQ condition). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Two-way ANOVA was used for the analysis of LC3B-II expression and Student’s t-test is used for the flux analysis. (d) The amount of SQSTM1 and TUBA in WT and mapk8−/- mapk9−/- immortalized MEFs after incubation with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. The SQSTM1:TUBA ratio was quantified and normalized to SQSTM1 expression in WT cells treated without torin 1 (mean ± SEM; n = 3 independent experiments; **, p < 0.01 (two-way ANOVA). (e) RPS6KB1, p-Thr389 RPS6KB1, AKT, p-Thr308 AKT, p-Ser473 AKT, and GAPDH expression by WT and mapk8−/- mapk9−/- immortalized MEFs after incubation (2 or 4 h) without or with 200 nM rapamycin was examined by immunoblot analysis. (f) LC3B and TUBA expression by WT and mapk8−/- mapk9−/- immortalized MEFs after incubation (2 h) without or with 200 nM rapamycin in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:TUBA ratios were normalized to the mean of WT control (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Two-way ANOVA was used for the analysis of LC3B-II expression and Student’s t-test was used for the flux analysis.

Fig 4: Schematic of cellular mechanisms of vasodilatation and protection of BRB integrity by IGFBP-3 via NO release.In endothelial cells, IGFBP-3 may directly activate the SRB1 or indirectly by releasing an unknown factor. Activation of SRB1 by IGFBP-3 activates eNOS via independent mechanisms. Ca2+-dependent pathway involves influx of Ca2+ via TRPV4 channels. Ca2+-calmodulin complex causes dephosporylation of Thr495 residue of eNOS resulting in the dissociation of the activated eNOS from caveolae however, this did not occur in microvascular endothelial cells. Ca2+-independent pathway involves activation of PI3K most likely by cSrc-kinase; PI3K activates Akt, which in turn activates eNOS by phosphorylation at Ser1177. NO released by IGFBP-3 mediates protective effect on BRB and vasodilatory functions by the PI3K/AKT pathway.

Fig 5: DD-Akt(E40K) induction is dose-responsive to TMP treatment and reversible.(A) Western immunoblotting analysis of dose-response of DD-Akt(E40K) induction by TMP. HEK293 cells were transfected with eGFP-only expressing vector (EV) or DD-Akt(E40K) fusion protein expression vector and treated with indicated doses of TMP for 24 hr before analysis by western immunoblotting with the indicated probes. (B) Quantification of DD-Akt(E40K) induction (as determined by western immunoblotting) by various doses of TMP. Values means with SEM. N = 3–4 independent experiments. (C) Time course of DD-Akt(E40K) induction by TMP. HEK293 cells were transfected with DD-Akt(E40K) expression vector, treated with 10 μM TMP for the indicated times and then assessed for total DD-Akt(E40K) levels by western immunoblotting as in (A). Values are means with SEM. N = 2–4 independent experiments. (D) Reversal of DD-Akt(E40K) induction after TMP washout. HEK293 cultures transfected with DD-Akt(E40K) as above were treated with 10 μM TMP for 24 hr, washed free of TMP and harvested for western immunoblotting at the indicated time points. Total DD-Akt(E40K) levels were determined by western immunoblotting as in (A). Data shown are from one experiment.