Fig 2: cAMP- and cGMP-dependent kinases activate mTORC1 signaling in adipocytes. (A) mTORC1 activity, measured by phosphorylation of S6K1 and S6, in Bat8 adipocytes, an immortalized mouse brown adipocyte cell line, in response to insulin (Ins) or isoproterenol (ISO) in the absence or presence of rapamycin (RAPA). (B) Bat8 adipocytes treated with ISO or 8-Br-cAMP with or without RAPA. (C) mTORC1 activity in hMADS, a human immortalized adipocyte cell line, in response to ANP, 8-Br-cAMP, or insulin, in the absence or presence of the PKA inhibitor, KT5720. (D) hMADS cells treated with ANP or 8-Br-cGMP in the absence or presence of RAPA. ANP treatment increases phosphorylation of VASP at Ser239, an established PKG phosphorylation site.

Fig 3: ENAH EVH1 binds to a peptide from photoreceptor cilium actin regulator (PCARE) differently than to the peptide FPPPP.(A) Surface representation of ENAH EVH1 bound to a segment of PCARE, highlighting Ena/VASP EVH1 domain conservation using colors as in Figure 1. (B) View comparing the orientations of an FP4 peptide and the LP4 region of PCARE. Side chains of the ENAH EVH1 domain are shown as sticks using tan for the FP4 complex and gray for the PCARE complex. (C) Surface representation of ENAH EVH1 domain bound to the PCARE peptide. The LP4 residues are light blue and other EVH1-interacting residues are green; insets show details of the interactions. Note that the PCARE828–848 peptide is numbered as 133–153 in the PDB file.

Fig 4: N-terminal phosphorylation of cGKI in purified preparations (A) and cell extracts (B).(A) Purified cGKIα or (B) cell extracts prepared from wild-type MEFs in non-denaturating buffer were incubated for 15 min at 30°C under control conditions (Ctr) or in the presence of 0.1 mM ATP or 0.1 mM ATP combined with 0.1 mM cGMP (cG/ATP). Alternatively, samples were pre-incubated for 15 min at 30°C with 0.1 mM cGMP. Then they were further incubated either under control conditions without ATP (cG pre) or in the presence of 0.1 mM ATP (cG pre+ATP) for another 15 min at 30°C. Purified proteins (20 ng) or cell extracts (10 µg) were analyzed for N-terminal phosphorylation of cGKIα by Western blotting with antisera AffPS3 and PS6. The total amount of cGKI was detected with a pan-(nonphospho-specific) cGKI antibody, and phospho-VASP in cell extracts was monitored with anti-VASP antibody. GAPDH was used as loading control for cell extracts. Below the Western blots, the semiquantitative densitometric analysis of phospho-cGKI signals is shown. It was performed using ImageJ software [37] and is given as the ratio of the intensity of the phospho-band detected by AffPS3 or PS6 (p-cGKI) divided by the intensity of the respective cGKI band detected by the pan-cGKI antibody in the same sample (cGKI). Data shown in the bar graphs in (A) are means ± SEM (n = 3 independent experiments); *p≤0.05, ***p≤0.001. Data shown in the bar graphs in (B) are means of 2 independent experiments.

Fig 5: Inhibiting autophagic degradation of Cav-1 could maintain LSECs fenestrae.Primary rats LSECs, isolated from rats and cultured in vitro, were treated with autophagy inhibitors (3MA or bafilomycin) for 3 days. a Red or yellow represents autolysosomes or autophagosomes, respectively, visualized by confocal microscopy. Quantification of autophagic flux (%) in 100 cells was analyzed. *P < 0.05 versus the autolysosomes of the control group; #P < 0.05 versus the autophagosomes of the control group. b Protein levels of LC3 II/I, Cav-1, p-eNOS (Ser1177), eNOS, p-VASP (Ser157), and VASP in primary LSECs analyzed by western blot. The relative protein expression is quantified in the graph, below. *P < 0.05 versus LC3 II/I of the control group; #P < 0.05 versus Cav-1/β-actin of the control group; &P < 0.05 versus p-eNOS/eNOS of the control group; $P < 0.05 versus p-VASP/VASP of the control group. c Interaction of LC3 with Cav-1 was detected by the co-IP assay. LC3 of primary LSECs was individually immunoprecipitated and Cav-1 subjected to immunoblotting analysis as indicated. d The co-localization of LC3 (green) with Cav-1 (red) and F-actin (blue) in LSECs of the three groups (CTR, 3MA, or bafilomycin), shown by immunofluorescence. Scale bar: 10 μm. e Magnification of SEM of LSECs in the three groups (CTR, 3MA, or bafilomycin) on Day 3, revealing the fenestrae structures (scale bar: 5 μm), and quantification of the total fenestral diameter, right. The black triangles indicate LSECs fenestrae structures. *P < 0.05 versus the control group. BA bafilomycin