Journal: Cell Communication and Signaling : CCS

Article Title: Activation of cannabinoid receptor type 2-induced osteogenic differentiation involves autophagy induction and p62-mediated Nrf2 deactivation

doi: 10.1186/s12964-020-0512-6

Figure Lengend Snippet: CNR2 agonists-induced osteogenic differentiation is blocked by autophagy inhibitor 3-MA. hFOB 1.19 cells were cultured at 34 °C until reaching confluence, and transferred to 39 °C. These cells were then treated with 2 mM 3-MA, 50 nM HU308 or 10 μM JWH133 for a - c 96 h or d 192 h. a The ALP activity was determined, and shown as mean ± standard deviation. The mRNA and protein levels of osteocalcin and osteopontin were determined with b real-time RT PCR and c western blotting, respectively. Data were shown as mean ± standard deviation. Cell mineralization was determined with Alizarin red staining. Heavier staining indicated stronger mineralization

Article Snippet: After being cultured at 39 °C for 48 h, cells were further treated with HU308 (5, 10, 25, 50 or 100 nM; purity ≥98% determined with HPLC; Tocris Bioscience, Minneapolis, MN, USA) or JWH133 (1, 2, 5, 10 or 20 μM; purity ≥98% determined with HPLC; Tocris Bioscience) for additional 12 h, 96 h or 192 h. As 100 nM HU308 and 20 μM JWH133 contained 0.05% DMSO, to eliminate the effects induced by the solvent, all cell culture media contained 0.05% DMSO.

Techniques: Cell Culture, Activity Assay, Standard Deviation, Quantitative RT-PCR, Western Blot, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Activation of cannabinoid receptor type 2-induced osteogenic differentiation involves autophagy induction and p62-mediated Nrf2 deactivation

doi: 10.1186/s12964-020-0512-6

Figure Lengend Snippet: CNR2 agonists activate autophagy and suppress mTOR pathway in hFOB 1.19 cells. hFOB 1.19 cells were cultured at 34 °C until reaching confluence, and then transferred to 39 °C. Forty-eight hours later, HU308 (5, 10, 25, 50 or 100 nM) or JWH133 (1, 2, 5, 10 or 20 μM) was added into the cell medium. Twelve hours later, hFOB 1.19 cells were subjected to a - b western blotting analysis. c For immunofluorescence staining of p62, hFOB 1.19 cells were treated with 50 nM HU308 or 10 μM JWH133 for 12 h. d - g hFOB 1.19 cells were infected with LV-CNR2 shRNA at 34 °C, and 24 h later, cells were transferred to 39 °C for differentiation. Forty-eight hours later, cells were further treated with 50 nM HU308 or 10 μM JWH133 for 12 h, and then harvested for western blotting analysis

Article Snippet: After being cultured at 39 °C for 48 h, cells were further treated with HU308 (5, 10, 25, 50 or 100 nM; purity ≥98% determined with HPLC; Tocris Bioscience, Minneapolis, MN, USA) or JWH133 (1, 2, 5, 10 or 20 μM; purity ≥98% determined with HPLC; Tocris Bioscience) for additional 12 h, 96 h or 192 h. As 100 nM HU308 and 20 μM JWH133 contained 0.05% DMSO, to eliminate the effects induced by the solvent, all cell culture media contained 0.05% DMSO.

Techniques: Cell Culture, Western Blot, Immunofluorescence, Staining, Infection, shRNA

Journal: Cell Communication and Signaling : CCS

Article Title: Activation of cannabinoid receptor type 2-induced osteogenic differentiation involves autophagy induction and p62-mediated Nrf2 deactivation

doi: 10.1186/s12964-020-0512-6

Figure Lengend Snippet: CNR2 agonists inhibit nuclear Nrf2 accumulation during hFOB 1.19 cell osteogenic differentiation. hFOB 1.19 cells were cultured at 34 °C until reaching confluence, and then transferred to 39 °C. Forty-eight hours later, HU308 (5, 10, 25, 50 or 100 nM) or JWH133 (1, 2, 5, 10 or 20 μM) was added into the cell medium. Twelve hours later, hFOB 1.19 cells were subjected to a - b western blotting analysis. c For immunofluorescence staining of Nrf2, hFOB 1.19 cells were treated with 50 nM HU308 or 10 μM JWH133 for 12 h. White arrows indicated cells positive to nuclear Nrf2, yellow arrows indicated cells negative to nuclear Nrf2. d - e hFOB 1.19 cells were infected with LV-CNR2 shRNA at 34 °C, and 24 h later, cells were transferred to 39 °C for differentiation. Forty-eight hours later, cells were further treated with 50 nM HU308 or 10 μM JWH133 for 12 h, and then harvested for western blotting analysis

Article Snippet: After being cultured at 39 °C for 48 h, cells were further treated with HU308 (5, 10, 25, 50 or 100 nM; purity ≥98% determined with HPLC; Tocris Bioscience, Minneapolis, MN, USA) or JWH133 (1, 2, 5, 10 or 20 μM; purity ≥98% determined with HPLC; Tocris Bioscience) for additional 12 h, 96 h or 192 h. As 100 nM HU308 and 20 μM JWH133 contained 0.05% DMSO, to eliminate the effects induced by the solvent, all cell culture media contained 0.05% DMSO.

Techniques: Cell Culture, Western Blot, Immunofluorescence, Staining, Infection, shRNA

Journal: Cell Communication and Signaling : CCS

Article Title: Activation of cannabinoid receptor type 2-induced osteogenic differentiation involves autophagy induction and p62-mediated Nrf2 deactivation

doi: 10.1186/s12964-020-0512-6

Figure Lengend Snippet: p62 stabilizes Nrf2 in hFOB 1.19 cells exposed to CNR2 agonists. hFOB 1.19 cells were cultured at 34 °C until reaching confluence and then transfected with empty vector or p62 overexpression vector (SQSTM1 OE). Then, culture medium was replaced, and cells were then cultured at 39 °C in presence of 50 nM HU308, 10 μM JWH133 for 48 h. a - b The protein levels of p62 and Keap1 were analyzed with western blotting. c Post a 48-h transfection, hFOB 1.19 cells were harvested at 0.5, 1, 3, 6, 8 h following treatment of 100 μg/mL cycloheximide. The expression levels of Nrf2 were determined with western blotting analysis. d - e For Co-IP assay, beads coated with anti-Keap1 was used to pull down intracellular Keap1, and the expression levels of Keap1 and Nrf2 were determined with western blotting

Article Snippet: After being cultured at 39 °C for 48 h, cells were further treated with HU308 (5, 10, 25, 50 or 100 nM; purity ≥98% determined with HPLC; Tocris Bioscience, Minneapolis, MN, USA) or JWH133 (1, 2, 5, 10 or 20 μM; purity ≥98% determined with HPLC; Tocris Bioscience) for additional 12 h, 96 h or 192 h. As 100 nM HU308 and 20 μM JWH133 contained 0.05% DMSO, to eliminate the effects induced by the solvent, all cell culture media contained 0.05% DMSO.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Over Expression, Western Blot, Expressing, Co-Immunoprecipitation Assay

Journal: Traffic (Copenhagen, Denmark)

Article Title: Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

doi: 10.1111/j.1600-0854.2005.00308.x

Figure Lengend Snippet: Figure 1: Endogenous JFC1 is phosphorylated via the phospha- tidylinositol 3-kinase/Akt pathway in vivo. A) Phosphatase and tensin homolog-null LNCaP cells were incubated in the presence of [32P]-Pi for 3 h at 37 C. Where indicated, cells were treated with 20 mM LY294002 for 3 h previous to the addition of 10 ng/ml interleukin-6 (IL-6). The cells were lysed and the activation of Akt was determined in these samples using an antibody that detects only the active kinase phosphorylated in position S473. Immunoprecipitation (IP) reactions using an antibody that specifically recognizes JFC1 were performed as described under Materials and Methods. Samples were resolved by gel electrophoresis, transferred tonitrocelluloseandanalyzedbyimmunoblotting[Western blot (WB)] for the presence of JFC1 in the immuno-pellets. The level of phosphorylation was detected by autoradiography. The signals for JFC1 and phosphorylated-JFC1 were quantified using the QUANTITY ONE 4.2.1 quantification software (Bio-Rad) and the ratio of JFC1-phosphorylation/JFC1 signal was calculated after densitometry detection (A, lower panel). Results are representa- tive of two independent experiments. B) Hela S3 cells were transfected with an expression vector that encodes a constitu- tively active form of Akt (pmyc-Akt-active) and/or the JFC1 expression vector (pBKCMV-JFC1) or the corresponding empty vector (–), according to the description in the figure and as described under Materials and Methods. Forty-eight hours after transfection, cells were incubated in the presence of [33P]-Pi for 4 h at 37 C. Cells were washed in PBS, harvested in RIPA buffer, and JFC1 was immunoprecipitated as described above. Immuno-pellets were resuspended in sample buffer, resolved by SDS–PAGE, transferred to nitrocellulose and JFC1 was immuno-detected (WB) before autoradiography. The level of expression of active Akt was evaluated using an antibody raised against the myc tag (Input).

Article Snippet: The anti-phosphorylated Akt (Thr-308) and the anti-phosphorylated Akt-substrate antibody were from cell signaling.

Techniques: In Vivo, Incubation, Activation Assay, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Phospho-proteomics, Autoradiography, Software, Transfection, Expressing, Plasmid Preparation, SDS Page

Journal: Traffic (Copenhagen, Denmark)

Article Title: Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

doi: 10.1111/j.1600-0854.2005.00308.x

Figure Lengend Snippet: Figure 4: In vivo and in vitro Akt-phosphorylated JFC1 behave in a similar fashion under 2D electrophoresis analysis. A) To examine JFC1 phosphorylation by 2D electrophoresis, Hela S3 cells (panels i–iii) were transfected with the JFC1 expression vector pCMV- JFC1 or the corresponding empty vector pCMV (pBKCMV, Stratagene) using FuGene 6 as indicated in the figure. After 48 h, cells were stimulated with 40 nM calyculin A or carrier (dimethyl sulphoxide) for 30 min at 37 C in the presence or absence of 20 mM LY294002. The cells were resuspended and lysed in sample rehydration buffer I (Invitrogen) containing 8 M Urea, 2% CHAPS and anti-phosphatases cocktail I and II (Calbiochem), and cellular proteins were further diluted in sample rehydration buffer II containing 8 M Urea, 2% CHAPS, 0.5% ZOOM carrier ampholytes (Invitrogen) and 0.002% bromophenol blue. Proteins (200 mg) were resolved by 2D electrophoresis using the ZOOM IPGRunner System and ZOOM Strips, pH 3–10 (Invitrogen) as described by the manufacturer. Gels were transferred to nitrocellulose, and proteins were detected by Western blot using an anti-phosphorylated Akt-substrate antibody (cell signaling). In panel I, the main spots representing Akt-phosphorylated proteins of around 62 kDa are indicated by arrows and arbitrarily denominated 1–5. In panel (ii), in the absence of JFC1, the loss of spots 2 and 3 is evident. These spots showed a mobility of approximately 64 and 62 kDa, respectively, corresponding to the mobility of the doublet frequently observed for JFC1 in gel electrophoresis (compare to panel iv).* indicates a major putative Akt substrate of around 40 kDa that decreases when JFC1 is expressed (compare to panel i), with unknown significance. In panel (iv), recombinant GST-JFC1 was phosphorylated with active Akt in vitro, separated from the kinase using GSH- beads, separated from the GST tag by digestion with PreScission Protease (Amersham Pharmacia Biotech) and resolved by 2D electrophoresis as described above. Akt-phosphorylated JFC1 was transferred to nitrocellulose and detected using an anti-phosphorylated Akt-substrate antibody. B) The anti-phosphorylated Akt-substrate antibody used in A, does not recognize unphosphorylated JFC1 (lane 1) but specifically recognizes Akt phosphorylated recombinant JFC1 by immunoblotting (WB) (lane 2).

Article Snippet: The anti-phosphorylated Akt (Thr-308) and the anti-phosphorylated Akt-substrate antibody were from cell signaling.

Techniques: In Vivo, In Vitro, Two-Dimensional Gel Electrophoresis, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Western Blot, Nucleic Acid Electrophoresis, Recombinant

Journal: Traffic (Copenhagen, Denmark)

Article Title: Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

doi: 10.1111/j.1600-0854.2005.00308.x

Figure Lengend Snippet: Figure 5: A) Akt-dependent phosphorylation sites identified in JFC1 by microcapillary high-performance liquid chromatography (HPLC) tandem mass spectrometry. Akt-dependent phosphorylated residues in JFC1 were detected using microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry and targeted ion MS/MS as described under Materials and Methods. The left panel shows a schematic representation of JFC1. The Rab-binding domain (RBD), containing the SHD domains responsible for the association with Rab27a, is shown in grey and the tandem C2-domains are shown in black. The numbers corresponding to the residues found to be phosphorylated by Akt. Five phosphopeptide sequences were detected by LC-MS/MS. The extended sequences surrounding these sites are shown in the right panel where basic residues are in bold and ‘p’ is preceding the phosphorylated site. B) Phosphatase and tensin homolog-null LNCaP prostate carcinoma cells (4 106 cells/condition) were transfected by nucleofection with 5 mg of a myc-tagged JFC1 expression vector, the indicated JFC1 mutant lacking a phosphorylated site or the empty vector (pCMV-Tag3) and 5 mg of the myc-Akt active expression vector or the corresponding empty vector (pUSE). Cells were seeded in 9-cm diameter plates and recovered in RPMI- 1640. After 48 h, cells were treated with LY294002 (20 mM) or buffer for 1 h at 37 C. Then, the medium was removed and replaced with 5 ml of phosphate-free medium containing 40 mM Hepes, pH 7.4 and 0.5 mCi of [32P]-Pi. After 1-h incubation at 37 C, the cells were scraped off, washed with PBS and resuspended in 500 ml of radio immunoprecipitation (IP) assay buffer. Cells were lysed on ice for 30 min to 1 h, spun down to eliminate debris, transferred to a CoIP spin column (Pierce), and 40 ml of anti-myc-agarose (Sigma-Aldrich) were added to the samples. Immunoprecipitation was carried out at 4 C overnight. Protein was eluted with sample buffer, resolved by gel electrophoresis, transferred to nitrocellulose and detected using an anti-myc monoclonal antibody (Cell signaling) [Western blot (WB)]. After detection of the immunoprecipitated protein, the membranes were immediately exposed for autoradiography. The arrows indicate the phosphorylation level of wild-type-JFC1 (WT) and JFC1-S241A in the presence of active Akt and in the absence of LY294002. C) The signals for JFC1 or JFC1-S241A in the WB and the signals for phosphorylated JFC1 or phosphorylated JFC1-S241A were quantified using the QUANTITY ONE 4.2.1 quantification software (Bio-Rad), and the ratio of JFC1-phosphorylation/JFC1 signal was calculated after densitometry detection. The results are mean SE of three different experiments. *p < 0.002.

Article Snippet: The anti-phosphorylated Akt (Thr-308) and the anti-phosphorylated Akt-substrate antibody were from cell signaling.

Techniques: Phospho-proteomics, High Performance Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Incubation, Radio Immunoprecipitation, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Autoradiography, Software

Journal: Traffic (Copenhagen, Denmark)

Article Title: Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

doi: 10.1111/j.1600-0854.2005.00308.x

Figure Lengend Snippet: Figure 8: Akt phosphorylation does not affect the ability of JFC1 to bind Rab27a. In vitro translated [S35]-Rab27a was pro- duced as described under Materials and Methods and loaded with the indicated nucleotides. GST-JFC1 or GST (negative control) were phosphorylated in vitro by Akt as described for Figure 3 and incubated in the presence of Rab27a for 12 h at 4 C. Recombinant GST-JFC1 or GST was pulled-down using GSH- agarose beads. The proteins in the pull-down were resuspended in sample buffer, resolved by gel electrophoresis and visualized by autoradiography. An aliquot of the phosphorylated protein was resolved by gel electrophoresis as control of phosphorylation (not shown).

Article Snippet: The anti-phosphorylated Akt (Thr-308) and the anti-phosphorylated Akt-substrate antibody were from cell signaling.

Techniques: Phospho-proteomics, In Vitro, Negative Control, Incubation, Recombinant, Nucleic Acid Electrophoresis, Autoradiography, Control

Journal: Natural Product Communications

Article Title: Convallatoxin Inhibits Cell Proliferation and Induces Cell Apoptosis by Attenuating the Akt-E2F1 Signaling Pathway in K562 Cells

doi: 10.1177/1934578x221136929

Figure Lengend Snippet: Figure 5. Convallatoxin inhibits the akt-E2F1 signaling pathway in K562 cells. (A) K562 cells were treated with different concentrations of convallatoxin for 24 h, and Western blot was performed to detect the expression of Akt-P308, total Akt and E2F1. (B-C) Gray value analysis was performed for each band. (D) Molecular structure of convallatoxin. (E) Proposed binding mode of convallatoxin (gold) in Akt1 (blue). Akt1 is shown in blue, while hydrogen bonds and hydrophobic force interactions are shown as blue solid lines and gray dashed lines, respectively. *P < .05, **P < .01, and ***P < .001 compared with the 0 μM convallatoxin group.

Article Snippet: IMDM medium, trypsin, penicillin, and streptomycin were purchased from Gibco| Life Technologies, fetal bovine serum (FBS) from Sijiqing Biological Engineering Materials Co. Ltd, dimethyl sulfoxide (DMSO), and enhanced chemiluminescence substrate ECL from Thermo Fisher Scientific, MMP detection kit (JC-1 method), lipofectamine 2000 kit, CCK-8 solution, PBS buffer, 4% tissue cell fixative, 1 mol/LTris-HCl buffer (pH= 6.8), and 10% SDS from Beyotime Biotechnology, primary antibodies against cleaved caspase-9, cleaved caspase-3, Bcl-2, Bax, p-Akt (Ser 308), t-Akt, E2F1, and GAPDH from Cell Signaling Technologies, Annexin V-FITC apoptosis assay kit and cell cycle analysis kit from MultiSciences (Lianke) Biotech Co., Ltd, E2F1 overexpression lentiviral plasmid and empty vector lentiviral plasmid from Jikai Gene Chemical Technology Co., Ltd, and convallatoxin (purity: analyzed by SDS-PAGE gel and HPLC, greater than 98%) from Sigma-Aldrich.

Techniques: Western Blot, Expressing, Binding Assay

Journal: Natural Product Communications

Article Title: Convallatoxin Inhibits Cell Proliferation and Induces Cell Apoptosis by Attenuating the Akt-E2F1 Signaling Pathway in K562 Cells

doi: 10.1177/1934578x221136929

Figure Lengend Snippet: Figure 6. E2F1 overexpression rescues the effects of convallatoxin in K562 cells. (A) Protein expression of E2F1 in K562 cells overexpressing the E2F1 gene was verified by Western blot. (B) K562 cells overexpressing E2F1 were treated with 10 μM of convallatoxin for 24 h, and cell viability was detected by CCK-8 assay. (C) Cell apoptosis was detected by Annexin V-FITC/PI staining. (D) Apoptosis rate is calculated as follows: number of apoptotic cells/total number of cells × 100%. **P < .01 and ***P < .001.

Article Snippet: IMDM medium, trypsin, penicillin, and streptomycin were purchased from Gibco| Life Technologies, fetal bovine serum (FBS) from Sijiqing Biological Engineering Materials Co. Ltd, dimethyl sulfoxide (DMSO), and enhanced chemiluminescence substrate ECL from Thermo Fisher Scientific, MMP detection kit (JC-1 method), lipofectamine 2000 kit, CCK-8 solution, PBS buffer, 4% tissue cell fixative, 1 mol/LTris-HCl buffer (pH= 6.8), and 10% SDS from Beyotime Biotechnology, primary antibodies against cleaved caspase-9, cleaved caspase-3, Bcl-2, Bax, p-Akt (Ser 308), t-Akt, E2F1, and GAPDH from Cell Signaling Technologies, Annexin V-FITC apoptosis assay kit and cell cycle analysis kit from MultiSciences (Lianke) Biotech Co., Ltd, E2F1 overexpression lentiviral plasmid and empty vector lentiviral plasmid from Jikai Gene Chemical Technology Co., Ltd, and convallatoxin (purity: analyzed by SDS-PAGE gel and HPLC, greater than 98%) from Sigma-Aldrich.

Techniques: Over Expression, Expressing, Western Blot, CCK-8 Assay, Staining