Fig 2: ULK1 interacts with PDK1 and induces phosphorylation. (A) HEK293T cells were transfected with HA-ULK1 and Myc-PDK1 constructs. After 48 h of transfection, the cells were lysed, and the lysates were subjected to immunoprecipitation using anti-Myc antibody. The immune complexes were washed and the immunoblots for PDK1 were performed as described in Materials and Methods (upper panel). The same cell lysates were used for anti-HA immunoblot (lower panel). (B) HA-ULK1 was expressed in HEK293T cells. Whole cell lysate was analyzed by immunoblotting for the indicated proteins. (C) HEK293T cells transfected with Myc-PDK1 and HA-ULK1 wild-type (WT) or kinase-inactive (KI) mutant were lysed and subject to immunoprecipitation with anti-Myc antibody. The immune complexes were incubated with ? phosphatase, washed, and subjected to anti-Myc immunoblot as described in Materials and Methods (top panel). Anti-Myc and anti-HA immunoblots were conducted using the same cell lysates (middle and bottom panels, respectively). (D) HA-ULK1 and GST-PDK1 were expressed in the HEK293T cells. The cell lysates were subjected to immunoprecipitation using anti-HA antibody. Immunoblots were obtained with the indicated antibodies. (E) The scheme of PDK1 truncation mutants. (F) HEK293T cells transfected with HA-ULK1 and GST-PDK1 WT, kinase and linker (KL), C-terminal (C) or PH domain (PH) constructs were lysed, and the lysates were subjected to co-immunoprecipitation. The results shown are representative of three independent experiments.

Fig 3: Nuclear FoxO1 levels and ESM-1 expression were regulated by PKC, PDK1 and PAK1 in RT-R-MDA-MB-231 cells. (A–C) RT-R-MDA-MB-231 cells were treated with GF109230X (100 nM), OSU03012 (1 µM) and FRAX1036 (5 µM) for the indicated times, and nuclear/cytosolic protein was extracted from the cells. FoxO1 protein expression levels in the cytosol or nucleus were analyzed by Western blotting. ß-Actin and PCNA were used as loading controls. The values are presented as the mean ± SD of five independent experiments. * p < 0.05, ** p < 0.01 compared to the control. CE, cytosolic extract; NE, nuclear extract. (D,E) MDA-MB-231 and RT-R-MDA-MB-231 cells were treated with the indicated inhibitors for 8 h and 16 h before collection of total RNA and protein, respectively. ESM-1 and GAPDH mRNA levels were analyzed by RT–PCR. ESM-1 and ß-actin protein levels were analyzed by Western blotting. The values are presented as the mean ± SD of five independent experiments. ** p < 0.01 compared to the MDA-MB-231 cells; # p < 0.05, ## p < 0.01 compared to the untreated RT-R-MDA-MB-231 cells.

Fig 4: 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 5: Overexpression of miR-16-2-3p suppresses MPMC proliferation and induces cell cycle arrest at G1 phase. Induction of miR-16-2-3p by pre16-2-3p transfection and relative expression levels of (A) miR-16-2-3p and (B) PDPK1 were determined by reverse transcription quantitative polymerase chain reaction. (C) MTT assay was performed to measure cell proliferation. (D) The number of Edu-stained cells was significantly decreased following pre16-2-3p transfection (magnification, x400). (E) Effect of miR-16-2-3p overexpression on cell cycle in MPMCs. Independent experiments were repeated at least 3 times, and the values are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. preNC. miRNA, microRNA; MPMCs, maxillary primordium mesenchymal cells; preNC, precursor negative control; pre16-2-3p, precursor miR-16-2-3p; PDPK1, 3-phosphoinositide-dependent protein kinase-1; Edu, 5-ethynyl-2'-deoxyuridine; NC, negative control; OD, optical density.