Journal: Journal of molecular and cellular cardiology

Article Title: PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis

doi: 10.1016/j.yjmcc.2014.02.007

Figure Lengend Snippet: REGγ controls PKAca stability. (A–B) REGγ negatively regulated PKAca level in multiple cell types. Primary HUVECs were treated with 20 nM siREGγ or a negative control (siNeg) (A, left panel). Primary REGγ+/+ and REGγ−/− MEFs from the third passage were used for Western blot analysis (A, right panel). Bone marrows (B, left panel) collected from day 14 REGγ+/+ and REGγ−/− mice were prepared for Western blot analysis. Relative PKAca levels were quantitated and normalized to β-actin as indicated by the numbers. (C) REGγ deficiency had no effect on PKAca mRNA levels. Total RNA extracted from primary HUVECs treated with 20 nM siREGγ or siNeg, primary MEFs, bone marrows, or spleens from REGγ+/+ and REGγ−/− mice, were prepared for real-time RT-PCR analysis. Data are shown as mean ± SD of three independent experiments. (D) REGγ dictated degradation kinetics of PKAca. Primary REGγ+/+ and REGγ−/− MEFs were treated with 100 µg/ml cycloheximide for different periods of time as indicated. Total cell lysates were extracted for Western blot (upper panel). Quantitated results of the Western blot analysis were plotted against indicated time course (lower panel). (E–F) Intracellular interactions between REGγ and PKAca. Immunoprecipitation was performed with whole cell lysates of HUVECs in the presence of MG132 using an anti-PKAca (E) or an anti-REGγ (F) antibody. The immuno-precipitated complexes were separated by SDS-PAGE and detected by an antibody against PKAca or REGγ.

Article Snippet: Cell lines and cell culture Primary HUVECs were purchased from ScienCell Research Laboratories and cultured in ECM (ScienCell, Carlsbad, CA, USA).

Techniques: Negative Control, Western Blot, Quantitative RT-PCR, Immunoprecipitation, SDS Page

Journal: Journal of molecular and cellular cardiology

Article Title: PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis

doi: 10.1016/j.yjmcc.2014.02.007

Figure Lengend Snippet: REGγ modulates phosphorylation and subcellular distribution of FoxO1. (A–B) REGγ depletion enhanced FoxO1 phosphorylation through activating PKA signaling. Immortalized REGγ+/− and REGγ−/− MEFs were serum-starved overnight and treated with DMSO, 20 uM forskolin (FSK) or 20 uM H89 for 30 min (A). Primary HUVECs were treated with/without siREGγ alone or in combination with siPKAca, then serum-starved overnight following incubating with 20 uM forskolin or DMSO for 30 min (B). Protein expression of endogenous p-FoxO1(Ser256), PKAca, FoxO1, REGγ, β-actin was detected by Western blot. (C) REGγ deficiency promoted FoxO1 nuclear export by activating PKA. Immunofluorescence staining was performed in REGγ+/− and REGγ−/− MEFs. Cells were serum-starved overnight before treatment with DMSO, 20 uM forskolin, or 20 uM H89, for 30 min. Cells were immunostained with anti-FoxO1 antibody (red) and the nuclei were counterstained with DAPI (blue). (D) The bar graph shows the corresponding quantitative analyses of the subcellular localization of FoxO1 and was generated by manually counting nuclear and cytoplasmic localization of FoxO1 in 400 FoxO1-positive staining MEFs for each condition. The area of the nucleus was determined using DAPI staining (nuclear staining). Data are shown as mean ± SD of quantitated results from three independent experiments (*p < 0.05 relative to DMSO-treated REGγ+/− MEFs; ★, p < 0.05 relative to DMSO-treated REGγ−/− MEFs).

Article Snippet: Cell lines and cell culture Primary HUVECs were purchased from ScienCell Research Laboratories and cultured in ECM (ScienCell, Carlsbad, CA, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Immunofluorescence, Staining, Generated

Journal: Journal of molecular and cellular cardiology

Article Title: PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis

doi: 10.1016/j.yjmcc.2014.02.007

Figure Lengend Snippet: REGγ tightly controls the expression of VCAM-1 and E-Selectin genes. (A) Cells lacking REGγ prohibited optimal expression of VCAM-1 and E-Selectin via activation of PKA in HUVECs. Total RNA extracted from primary HUVECs separately treated with control siNeg (lane 1), siREGγ (lane 2), siNeg + H89 (20 uM, 6 h) (lane 3), siREGγ + H89 (20 uM, 6 h) (lane 4), siNeg + siPKAca (lane 5) or siREGγ + siPKAca (lane 6), was prepared for real-time RT-PCR analysis. Cells were serum-starved overnight before H89 treatment. Statistical analyses were performed using the one-way ANOVA. Data are shown as mean ± SD of three independent experiments (*, p < 0.05;; ***, p < 0.001). (B) Depleted of REGγ prohibited optimal expression of VCAM-1 and E-Selectin via activation of PKA in HMEC-1 cells. HMEC-1 cells separately treated with 20 nM siNeg or siREGγ, following incubated with DMSO (lanes 1&4), 20 uM forskolin (lanes 2&5) or 20 uM H89 (lanes 3&6) for 6 h. Cells were serum-starved overnight before forskolin or H89 treatment. Total RNA extracted from HMEC-1 was prepared for real-time RT-PCR analysis. Statistical analyses were performed using the one-way ANOVA. Data are shown as mean ± SD of three independent experiments (**, p < 0.01; ***, p < 0.001). (C) FoxO1 is required for the expression of VCAM-1 and E-Selectin genes. Total RNA from primary HUVECs treated with 20nM siNeg or siFoxO1 for 48 h was prepared for real-time RT-PCR analysis. Cells were serum-starved overnight before RNA extraction. Data are shown as mean ± SD of three independent experiments (**, p < 0.01). (D) REGγ is responsible for VEGF-induced expression of VCAM-1 and E-Selectin genes. Primary HUVECs treated with 20 nM control siRNA (siNeg) were incubated with either vehicle (lane 1), 20uM forskolin (lane 2) for 6 h, 20 µg/ml VEGF (lane 3) for 2 h, or a combination of 20 uM forskolin (6 h) and 20 µg/ml VEGF treatment (2 h) (lane 4). HUVECs transfected with 20nM siREGγ were treated with 20ug/ml VEGF for 2 h (lane 5). Cells were serum-starved overnight before forskolin and VEGF treatment. Total RNA extracted from HUVECs was prepared for real-time RT-PCR analysis. Statistical analyses were performed using the one-way ANOVA. Data are shown as mean ± SD of three independent experiments (*, p < 0.05; ***, p < 0.001).

Article Snippet: Cell lines and cell culture Primary HUVECs were purchased from ScienCell Research Laboratories and cultured in ECM (ScienCell, Carlsbad, CA, USA).

Techniques: Expressing, Activation Assay, Control, Quantitative RT-PCR, Incubation, RNA Extraction, Transfection

Journal: Science Advances

Article Title: Cytosolic delivery of proteins by cholesterol tagging

doi: 10.1126/sciadv.abb0310

Figure Lengend Snippet: ( A ) Protein delivery in cell lines. ( B ) Protein delivery in stem cells and primary cells. The tagged aprotinin (molar ratio, 5:1) was incubated with cells for 2 hours at 37°C followed by counterstaining of the cell boundaries with WGA. Full names of the primary cells in (B) are human adipose–derived adult stem cells, primary human dermal fibroblasts, and primary HUVECs, respectively. Protein, aprotinin-AF488; WGA, WGA-AF594. Scale bar, 30 mm.

Article Snippet: The primary cells, including human adipose–derived stem cells, primary human dermal fibroblasts, and primary HUVECs, were purchased from Zen-Bio Inc. MCF-7 (ATCC #HTB-22), MDA-MB-231 (ATCC #HTB-26), PC-3 (ATCC #CRL-1687), SK-BR-3 (ATCC #HTB-30), and HeLa (ATCC #CCL-2) cells were cultivated in RPMI 1640 medium supplemented with fetal bovine serum (FBS; 10%), penicillin (100 U/ml), and streptomycin (100 μg/ml).

Techniques: Incubation, Derivative Assay