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: Communications Biology

Article Title: SARS-CoV-2 proteins and anti-COVID-19 drugs induce lytic reactivation of an oncogenic virus

doi: 10.1038/s42003-021-02220-z

Figure Lengend Snippet: a The iSLK.219 cells were transfected with vector control or vectors encoding SARS-CoV-2 spike protein (S), nucleocapsid protein (N) and KSHV RTA (as a positive control) with or without low dose of doxycycline (Dox, 0.1 µg/mL) induction for 72 h. The expression of RFP (representing viral lytic reactivation) and GFP (representing infected cells) were detected using fluorescence microscopy. Error bars represent S.D. for 4 image fields per well. b , c BCP-1 cells were transfected as above with or without low dose of 12- O -tetradecanoyl-phorbol-13-acetate (TPA, 1.0 ng/mL) induction for 72 h, then the transcripts of representative lytic genes were quantified by using qRT-PCR. The supernatants from transfected cells were collected to infect naive HEK293T cells, then viral genome levels were quantified by using qPCR with Lana -specific primers. Error bars represent S.D. for 3 independent experiments, ** p < 0.01 (vs the vector control). d Expression of ACE2 and LANA (as well as representative IgG control) in formalin-fixed paraffin-embedded KS tissues from 3 HIV+ patients and normal skin tissues were determined by immunohistochemical staining as described in the “Methods”. Bars: 50 μm.

Article Snippet: Human iSLK.219 cells were transfected with recombinant vectors of SARS-CoV-2 spike protein (S), nucleocapsid protein (N) (both purchased from Sino Biological), pCR3.1-RTA (a gift from Dr. Yan Yuan, University of Pennsylvania) , pFLAG-CMV2-p65 (pFLAG-p65, a gift from Dr. Ren Sun, University of California Los Angeles) , or vector controls, using Lipofectamine TM 3000 reagent (Invitrogen).

Techniques: Transfection, Plasmid Preparation, Positive Control, Expressing, Infection, Fluorescence, Microscopy, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded, Immunohistochemical staining, Staining

Journal: SLAS technology

Article Title: Simplifying Glycan Profiling through a High-Throughput Micropermethylation Strategy

doi: 10.1177/2472630320912929

Figure Lengend Snippet: Glycans were released from glycoproteins by PNGase F (N-glycans) or β-elimination (O-glycans), micropermethylated, cleaned up by C18 tips, and analyzed by MALDI-MS/ESI-MSn. MALDI-MS spectra of released and permethylated glycans: (A) N-glycans released from 5 million HEK 293f cells, (B) N-glycans released from 5 μL of human plasma, and (C) O-glycans released from 5 μL of human plasma. (D) Comparison of relative abundances of glycoforms detected from 10 micropermethylated reactions on N-glycans released from the same human serum IgG (no statistically significant difference between reactions [ANOVA, p > 0.05]). Maltoheptaose (after permethylation; m/z 1497.6 for N-glycans in nonreduced form and m/z 1513.8 for O-glycans in reduced form) is used as an internal standard for quantitation.

Article Snippet: Purified human serum IgG was purchased from Innovative Research (Novi, MI) (cat. 7660), and human plasma was purchased from Tennessee Blood Services (Memphis, TN).

Techniques: Quantitation Assay

Journal: SLAS technology

Article Title: Simplifying Glycan Profiling through a High-Throughput Micropermethylation Strategy

doi: 10.1177/2472630320912929

Figure Lengend Snippet: Relative Percentage of Permethylated Glycans from 10 Replicates of Micropermethylation Reactions on N-Glycans Released from Human Serum IgG.

Article Snippet: Purified human serum IgG was purchased from Innovative Research (Novi, MI) (cat. 7660), and human plasma was purchased from Tennessee Blood Services (Memphis, TN).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Extracellular vesicles as prospective biological indicators for midgestational placental complications in the mouse

doi: 10.3389/fcell.2025.1636335

Figure Lengend Snippet: The maternal-fetal interface of the Nodal ∆/∆ dams exhibits vascular development deficiencies, expansion of trophoblast giant cell layer and an altered gene expression profile at mid-pregnancy: (A, B) Immunofluorescence staining of sectioned implantation sites containing the maternal uterus, maternal decidua, and fetal placenta from the Nodal loxP/loxP and Nodal ∆/∆ mice at D8.5 with endothelial cell marker CD31 (red) and DAPI (blue). (B) Is higher magnification of the area marked by white rectangles in (A) . (C) Western blot and corresponding bar graph quantifying CD31 protein expression in the implantation sites from the Nodal loxP/loxP and Nodal ∆/∆ mice at D8.5. Values on the bar graph display the mean signal intensity of CD31 normalized against α-tubulin ± SD and *p < 0.05 indicates a significant difference. (D) Immunofluorescence staining of sectioned implantation sites from the Nodal loxP/loxP and Nodal ∆/∆ mice at D10.5 with trophoblast giant cell marker PL1 (green), CD31 (red) and DAPI (blue). Bottom images are a higher magnification of the area marked by white rectangles in the top images. (E) Immunofluorescence staining of sectioned implantation sites from the Nodal loxP/loxP and Nodal ∆/∆ mice at D10.5 with PL1 (green), spongiotrophoblast marker TPBPA (red) and DAPI (blue). (F) Bar graph illustrating the differential expression of various genes from the 84-gene Mouse Pre-Eclampsia RT 2 profiler array between the Nodal loxP/loxP and Nodal ∆/∆ mice at the D10.5 maternal-fetal interface where *p < 0.05, † p < 0.01, and ‡ p < 0.001 indicate significant differences. Scale bars represent 100 μm. Different layers within the implantation site are indicated as My, myometrium; MD, maternal decidua basalis; E, embryonic originated cells; DP, decidua parietalis. The orientation of the tissue is indicated by M, mesometrial; AM, anti-mesometrial.

Article Snippet: Slides were rinsed in permeabilization buffer twice (TBS, 0.2% BSA, 0.025%–0.25% Triton X-100) for 5 min, then incubated overnight at 4°C with primary antibodies for Placental lactogen I (PL1) (sc-34713, Santa Cruz, stock concentration 0.2 mg/mL, diluted 1:50 in 1% BSA/TBS), CD31 (ab182981, Abcam, stock concentration 0.519 mg/mL, diluted 1:1,500 in 1% BSA/TBS), or TPBPA (ab104401, Abcam, stock concentration 0.9 mg/mL, diluted 1:200 in 1% BSA/TBS).

Techniques: Gene Expression, Immunofluorescence, Staining, Marker, Western Blot, Expressing, Quantitative Proteomics