Fig 2: Total PDK1 levels and its phosphorylation are unaltered after synaptic activity and muscle contraction. PDK1 and pPDK1 in presynaptic stimulation treatment, Contraction treatment and Presynaptic stimulation with contraction treatment at 1 Hz stimulation for 30 min. Presynaptic stimulation has been simplified as Stimulation. Each column has been compared to its respective control (see Table 1). Results showed that PDK1 and pPDK1 levels, as well as pPDK1/PDK1 ratio, remained unaltered after nerve stimulation with and without muscle contraction. Data are mean percentage ± SEM, (Stimulation n = 7, Contraction n = 6 and Stimulation with Contraction n = 6). Abbreviations: PDK1, phosphoinositide-dependent kinase 1; pPDK1, phosphorylated phosphoinositide-dependent kinase 1.

Fig 3: Endothelial Gata5 is responsible for increased blood pressure.(a) GATA5 is expressed in human cardiac microvascular (CM), coronary artery (CA), dermal microvascular (DM) and pulmonary microvascular (PM) endothelial cells. (b,c) The vasoconstrictor response of Gata5-null mice mesenteric arteries to norepinephrine is unaltered (n=7 per group), while the vasodilatory response to acetylcholine is decreased (n=10–11 per group). The results are reported as mean±s.e.m. *P<0.05 versus controls (comparison of best-fit values—effector concentration for half-maximum response (EC50) and Hill slope—using an F-test). (d–f) Deletion of Gata5 in endothelial (eGata5-null mice; n=4–5 per group) but not smooth muscle cells (smGata5-null mice; n=5 per group) decreases mesenteric arteries sensitivity to acetylcholine and increases BP (eGata5-null mice n=6–10 per group; smGata5-null mice n=6–9 per group). The results are reported as mean±s.e.m. *P<0.05 versus controls (two-factor ANOVA). (g) The vasodilatory response of Gata5-null mice to diethylamine NONOate, an NO donor, is unaltered (n=7 per group). The results are reported as mean±s.e.m. (comparison of best-fit values—EC50 and Hill slope—using an F-test). (h,i) NOS3 and Akt phosphorylation are decreased in Gata5-null mice mesenteric arteries. PTEN and PDK1 phosphorylation and expression are unaltered (n=5–7 per group). Phosphorylated proteins are normalized to total proteins. Total proteins are normalized to actin. The results are reported as mean±s.e.m. *P<0.05 versus Gata5+/+ mice (t-test). (j) Quantification of protein nitrotyrosination in mesenteric arteries of Gata5-null mice and their controls as measured by ELISA. 3-Nitrotyrosine content is expressed as picomole of nitrotyrosine per milligram of protein (n=5–7 per group). The results are reported as mean±s.e.m. (t-test).

Fig 4: PIK3CA, PDK1 and pAKT alterations in human HNSCCs(a) qRT-PCR quantitation of PIK3CA mRNA expression in human HNSCCs. Error bars indicate mean±SD. *p<0.05. NN: Normal controls; Adj: adjacent mucosa; Well: well differentiated tumors; Mod: moderately differentiated tumors; Poorly: poorly differentiated tumors; LN mets: Lymph node metastases.(b) Percentage of human HNSCC cases which exhibited =2+ IHC staining of p110a, PDK1, and pAKT. Well: well differentiated tumors; Mod: moderately differentiated tumors; Poorly: poorly differentiated tumors; LN mets: Lymph node metastases. *p<0.05.(c) IHC staining of serial sections of a poorly differentiated HNSCC. Scale bar: 50 µm.

Fig 5: Overexpression and activation of PDK1 but not AKT in PIK3CA-GEMM tumors(a) Relative mRNA levels of PIK3CA, AKT1, AKT2, PDK1 and PTEN measured by qRT-PCR. Results are averages of triplicate experiment. *p<0.05 (n=6 for each group).(b) Western blotting analysis of tumor samples from control mice and PIK3CA-GEMMs using the antibodies as indicated.(c) Immunohistochemistry (IHC) staining of p110α, pAKTser473 and PDK1 in tumors from PIK3CA-GEMMs and control mice. Scale bar: 50 μM(d) qRT-PCR examination of mRNA levels of molecules commonly altered in human HNSCCs. Results are averages of triplicate experiments. *p<0.05 (n=6 for each group).(e) Relative mRNA levels of PDK1, E-cadherin, and Vimentin in CU110 cells with PDK1 knockdown (CU110-shPDK1) or with a scramble control (CU110-SCR) measured by qRT-PCR. *p<0.05.(f) Cell proliferation assay of CU110-shPDK1 and CU110-SCR measured by the Cell Counting Kit-8. *p<0.05.(g) Migration assay in CU110-shPDK1 and CU110-SCR cells. Dotted line defines the edges of cells. All experiments were performed in triplicate. *p<0.05.(h) Invasion assay in CU110-SHPDK1 and CU110-SCR cells. All experiments were performed in triplicate. *p<0.05.