Journal: Journal of cell science

Article Title: FGF2-induced Ras-MAPK signalling maintains lymphatic endothelial cell identity by upregulating endothelial-cell-specific gene expression and suppressing TGFβ signalling through Smad2.

doi: 10.1242/jcs.137836

Figure Lengend Snippet: Fig. 7. H-Ras overexpression inhibits TGFb1-induced Smad2 phosphorylation. (A) Western blots of C-terminally phosphorylated, linker phosphorylated and total Smad2 in control and H-Ras-overexpressing mLECs stimulated by TGFb1, and quantitative analysis of western blots. Error bars s.d.; n53. ***P,0.005 (versus TGFb1-stimulated control mLECs), **P,0.01 (versus starved control mLECs). (B) Western blots of phosphorylated and total Smad1/3/5 in control and H-Ras-overexpressing mLECs stimulated with TGFb1, and quantitative analysis of western blots. (C) Real-time RT-PCR assay for mRNA expression in control and H-Ras-overexpressing mLECs cultured in EGM2-MV CM and FGF2-depleted EGM2-MV CM supplemented with TGFb1 (CM-FGF2+TGFb). Error bars represent s.d.; n53. **P,0.01, ****P,0.001 (versus control mLECs cultured in CM).

Article Snippet: Western blot analysis was performed using the following primary antibodies: mouse anti-Pan-Ras (Calbiochem/Merck, Darmstadt, Germany), rabbit anti-phosphorylated p42/44 MAPK, anti-p42/44 MAPK, anti-phosphorylated Smad2 (Ser465/ 467; 138D4), anti-phosphorylated Smad2 (Ser245/250/255), antiphosphorylated Smad3 (Ser423/425; C25A9), anti-phosphorylated Smad1/5 (Ser463/465; 41D10), anti-Smad2 XP (D43B4), anti-Smad3 (C67H9), anti-Smad1 XP (D59D7) and anti-Smad5 (Cell Signaling Technology).

Techniques: Over Expression, Phospho-proteomics, Western Blot, Control, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Journal of cell science

Article Title: FGF2-induced Ras-MAPK signalling maintains lymphatic endothelial cell identity by upregulating endothelial-cell-specific gene expression and suppressing TGFβ signalling through Smad2.

doi: 10.1242/jcs.137836

Figure Lengend Snippet: Fig. 8. MAPKs repress Smad2 activation by phosphorylating the Smad2 linker region in the absence of TGFb1 and suppressing the TGFb1-driven phosphorylation of Smad2 C-terminal region. (A) Western blots of Smad2 and MAPK phosphorylation in mLECs stimulated with TGFb1 in the presence or absence of a MEK inhibitor U0126, and quantitative analysis of western blots. Error bars represent the s.d.; n53. *P,0.05, ****P,0.001. (B) Real-time RT-PCR assay for expression of Snai1 and Snai2 mRNA in mLECs cultured in EGM2-MV CM and TGFb1-supplemented EGM2-MV (CM+TGFb) in the presence or absence of U0126. Error bars represent the s.d.; n53. ****P,0.001. (C) Schematic representation of the role of FGF2 in EndMT of LECs. In LECs, the FGF2– FGFR–Ras–MAPK pathway not only maintains endothelial marker gene expression, but also induces phosphorylation of the Smad2 linker, leading to suppression of TGFb-induced Smad2 activation. FGF2 represses EndMT by maintaining endothelial marker gene expression and suppressing TGFb–Smad2- induced activation of mesenchymal genes.

Article Snippet: Western blot analysis was performed using the following primary antibodies: mouse anti-Pan-Ras (Calbiochem/Merck, Darmstadt, Germany), rabbit anti-phosphorylated p42/44 MAPK, anti-p42/44 MAPK, anti-phosphorylated Smad2 (Ser465/ 467; 138D4), anti-phosphorylated Smad2 (Ser245/250/255), antiphosphorylated Smad3 (Ser423/425; C25A9), anti-phosphorylated Smad1/5 (Ser463/465; 41D10), anti-Smad2 XP (D43B4), anti-Smad3 (C67H9), anti-Smad1 XP (D59D7) and anti-Smad5 (Cell Signaling Technology).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Quantitative RT-PCR, Expressing, Cell Culture, Marker, Gene Expression

Journal: AIDS Research and Therapy

Article Title: Quercetin ameliorates HIV-1 gp120 protein-induced intestinal barrier dysfunction by inhibiting the activation of the ERK1/2 signaling pathway in a Caco-2 cell model

doi: 10.1186/s12981-026-00846-9

Figure Lengend Snippet: Quercetin mitigates HIV-1 gp120-induced intestinal barrier dysfunction in Caco-2 cells. A Caco-2 cells were treated with 1-64 μM Quercetin for 24 h to evaluate cell viability compared no Quercetin.Values are mean ± SD (n = 5). B The effects of Quercetin and gp120 on Caco-2 cell viability were evaluated using the CCK-8 assay after 24 h. Values are mean ± SD (n = 5). C The permeability of Caco-2 cell monolayers was assessed by measuring TEER values at 0, 6, 12, and 24 h. Values are mean ± SD (n = 3). D FITC-dextran flux was measured after 24 h. Values are mean ± SD (n = 5). E , F Expression levels of ZO-1, Occludin, and Claudin-1 mRNA and protein were quantified using RT-qPCR and Western blot analysis after Caco-2 cell monolayers were treated with different drugs for 24 h. Values are mean ± SD (n = 3). G Immunofluorescence staining was used to visualize the localization and expression of ZO-1, Occludin, and Claudin-1 after Caco-2 cell monolayers were treated with different drugs for 24 h. Tight junction proteins (green) were labeled with fluorescent secondary antibodies, and nuclei (blue) were counterstained with DAPI (Scale bar: 150 μm). Values are mean ± SD (n = 3). H Flow cytometry analysis of Annexin V-FITC/PI-stained cells. Flow cytometry was performed to assess apoptosis in Caco-2 cells after Caco-2 cell monolayers were treated with different drugs for 24 h. Values are mean ± SD (n = 3). Data was normalized to control values. Statistical significance: * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: Fetal bovine serum (F8318) and lipopolysaccharide LPS (L4391), fluorescein isothiocyanate-labeled dextran (FITC-dextran, FD4) and Bovine Serum Albumin (A1933) were purchased from Sigma-Aldrich (St. Louis, MO, USA), recombinant human HIV-1 gp120 (CN54) protein (ab174070) and secondary anti-goat anti-rabbit IgG Alexa Fluor® 488 (ab150077) were purchased from Abcam (Cambridge, UK), Quercetin (DR10620) was purchased from Dingrui Chemical Co Ltd (Shanghai, China), 12-well transwell culture chambers (3401) (pore size 0.4 μm, diameter 12 mm, growth area 1.12 cm 2 ) were purchased from Corning (Cambridge, MA, USA), alkaline phosphatase kits (A059) were purchased from Nanjing Jianjian Bioengineering Institute (Nanjing, China), CCK-8 kits (C0043), Annexin V-FITC apoptosis detection kits (C1062), anti-fluorescence quencher (P0126), and 4′,6-diamidino-2-phenylindole (DAPI) (C1005) were purchased from Biyuntian Biotechnology (Shanghai, China), Total RNA Extraction Kit (9767), cDNA Reverse Transcription Kit (RR047A), and TB Green ® Premix Ex TaqTM II (RR820A) were purchased from Takara Bio (Shiga, Japan), and BCA Protein Analysis Kits (23,227) were purchased from Thermo Fisher Scientific (Burlington, ON, Canada), U0126 (9903), primary antibody ZO-1 (13,663), Occludin (91,131), Claudin-1 (13,255), phospho-p44/42 ERK1/2 ( 4370), p44/42 ERK1/2 (4695), phospho-MLC (3671), and MLC (8505) were purchased from Cell Signaling Technology (Danvers, MA, USA), MLCK (YA3133) was purchased from Med Chem Express (New Jersey, USA), β-actin (49,294) and Goat Anti-Rabbit IgG (H&L) HRP ( L35009 ) were purchased from Signalway Antibody (College Park, MD, USA), and TRIzol (411,308) was purchased from Ambion (Texas, USA).

Techniques: CCK-8 Assay, Permeability, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Labeling, Flow Cytometry, Control

Journal: AIDS Research and Therapy

Article Title: Quercetin ameliorates HIV-1 gp120 protein-induced intestinal barrier dysfunction by inhibiting the activation of the ERK1/2 signaling pathway in a Caco-2 cell model

doi: 10.1186/s12981-026-00846-9

Figure Lengend Snippet: Quercetin enhances DUSP family expression in HIV-1 gp120-stimulated Caco-2 cells, leading to MAPK signaling pathway inhibition. A Venn diagram of differentially expressed genes (DEGs). DEGs between the control group (A) and the 100 ng/mL gp120 group (B) are shown in green, while DEGs between the 100 ng/mL gp120 group (B) and the 100 ng/mL gp120 + 2 μM Quercetin group (C) are shown in orange. A total of 478 shared DEGs (brown) were identified. B KEGG enrichment analysis of shared DEGs in both comparison groups (B/A and C/B), highlighting the MAPK signaling pathway. Values are mean ± SD (n = 3). C RNA-seq-based heatmap illustrating DUSP family mRNA expression levels in the control, 100 ng/mL gp120, and 100 ng/mL gp120 + 2 μM Quercetin groups. The x-axis represents log₂. (expression value +1) , while the y-axis represents the gene. Red indicates higher expression levels, while blue represents lower expression levels. D RT-qPCR was performed to quantify DUSP4, DUSP5, and DUSP8 mRNA expression levels. Caco-2 cell monolayers were treated with or without 10 μM U0126 and 2 μM Quercetin in the presence of 100 ng/mL gp120 for 24 h. Values are mean ± SD (n = 3). E Western blot analysis of ERK1/2 phosphorylation levels. Caco-2 cell monolayers were treated with or without 10 μM U0126 and 2 μM Quercetin in the presence of 100 ng/mL gp120 for 24 h. Phosphorylated protein levels were normalized to total protein content, with β-actin used as an internal control. Values are mean ± SD (n = 3). (F) TEER measurements of Caco-2 cell monolayers at 24 h. Values are mean ± SD (n = 3). G Western blot analysis of ZO-1, Occludin, and Claudin-1 protein expression. Caco-2 cell monolayers were treated with or without 10 μM U0126 and 2 μM Quercetin in the presence of 100 ng/mL gp120 for 24 h. Values are mean ± SD (n = 3). Data was normalized to control values. Statistical significance: * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: Fetal bovine serum (F8318) and lipopolysaccharide LPS (L4391), fluorescein isothiocyanate-labeled dextran (FITC-dextran, FD4) and Bovine Serum Albumin (A1933) were purchased from Sigma-Aldrich (St. Louis, MO, USA), recombinant human HIV-1 gp120 (CN54) protein (ab174070) and secondary anti-goat anti-rabbit IgG Alexa Fluor® 488 (ab150077) were purchased from Abcam (Cambridge, UK), Quercetin (DR10620) was purchased from Dingrui Chemical Co Ltd (Shanghai, China), 12-well transwell culture chambers (3401) (pore size 0.4 μm, diameter 12 mm, growth area 1.12 cm 2 ) were purchased from Corning (Cambridge, MA, USA), alkaline phosphatase kits (A059) were purchased from Nanjing Jianjian Bioengineering Institute (Nanjing, China), CCK-8 kits (C0043), Annexin V-FITC apoptosis detection kits (C1062), anti-fluorescence quencher (P0126), and 4′,6-diamidino-2-phenylindole (DAPI) (C1005) were purchased from Biyuntian Biotechnology (Shanghai, China), Total RNA Extraction Kit (9767), cDNA Reverse Transcription Kit (RR047A), and TB Green ® Premix Ex TaqTM II (RR820A) were purchased from Takara Bio (Shiga, Japan), and BCA Protein Analysis Kits (23,227) were purchased from Thermo Fisher Scientific (Burlington, ON, Canada), U0126 (9903), primary antibody ZO-1 (13,663), Occludin (91,131), Claudin-1 (13,255), phospho-p44/42 ERK1/2 ( 4370), p44/42 ERK1/2 (4695), phospho-MLC (3671), and MLC (8505) were purchased from Cell Signaling Technology (Danvers, MA, USA), MLCK (YA3133) was purchased from Med Chem Express (New Jersey, USA), β-actin (49,294) and Goat Anti-Rabbit IgG (H&L) HRP ( L35009 ) were purchased from Signalway Antibody (College Park, MD, USA), and TRIzol (411,308) was purchased from Ambion (Texas, USA).

Techniques: Expressing, Inhibition, Control, Comparison, RNA Sequencing, Quantitative RT-PCR, Western Blot, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: Lnk-dependent axis of SCF–cKit signal for osteogenesis in bone fracture healing

doi: 10.1084/jem.20100321

Figure Lengend Snippet: Enhanced SL cell mobilization by SCF stimulation in Lnk KO mice. (a) Serum mouse SCF levels were measured in Lnk KO and WT mice after fracture by ELISA. ††, P < 0.01; *, P < 0.05 versus Pre, respectively. (b) The number of circulating SL cells in Lnk KO and WT mice with injection of PBS (control; Lnk KO-PBS and WT-PBS), SCF (SCF, 20 µg/kg; Lnk KO-SCF and WT-SCF), or soluble SCF receptor (=c-kit ligand, sKit, 20 µg/kg; Lnk-sKit) were assessed by FACS in the indicated time course after the treatment. †† and †, P < 0.01 and P < 0.05 versus Pre, respectively. ** and *, P < 0.01 and P < 0.05 versus WT-PBS, WT-SCF, Lnk KO-PBS, and Lnk KO-sKit, respectively. (c) The number of circulating SL cells in WT mice with fracture and injection of PBS, SCF (20 µg/kg), or SCF (20 µg/kg) + sKit (WT-fx-PBS, WT-fx-SCF, or WT-fx-SCF-sKit, respectively), WT mice without fracture and SCF (20 µg/kg; WT-SCF), and Lnk KO mice with fracture and sKit (Lnk KO-fx-sKit) were assessed by FACS in the indicated time course after the treatment. †† and †, P < 0.01 and P < 0.05 versus Pre, respectively. * and **, P < 0.05 and P < 0.01 versus WT-fx-SCF-sKit, respectively. (d) Immunofluorescent staining for CD31 (green) and OC (OC, green) were performed in granulation tissue samples of peri-fracture sites in Lnk KO (left one panel) and WT (right two panels) mice 7 d after fracture. Alcian blue/orange G stained sections were also shown in parallel with immunostained sections (lower panels to each immunofluorescent staining images). Mouse phenotype with SCF (20 µg/kg) or sKit (20 µg/kg) administration (+) is indicated in the lower part of images. Dotted line indicates bone surface. Bar = 50 µm. Quantification of capillaries (e) and OBs (f). CD31 positive capillaries and OC positive OBs were counted in 3 randomly selected high power fields and averaged.**, P < 0.01 and *, P < 0.05. All data averaged with SEM from five independent experiments. All experiments were obtained from triplicated assays.

Article Snippet: Purified recombinant human SCF and sKit were purchased from R&D Systems and supplemented concentrations of cytokines used for culture were as follows: SCF 100 ng/ml and sKit 10 μg/ml (Nakamura, 2004).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: Lnk-dependent axis of SCF–cKit signal for osteogenesis in bone fracture healing

doi: 10.1084/jem.20100321

Figure Lengend Snippet: Enhanced mineralization of OBs in Lnk-deficient mice. (a) OBs (OBs) isolated from Lnk KO and WT mice were cultured in osteogenic condition medium. Cells were assessed morphologically by ALP staining (blue) at day 14 and by alizarin red staining (red) at day 21. Upper panels show macroscopic images of whole culture dishes and lower panels show magnified images of nodule with mineralized matrix formation. (b) Real-time RT-PCR of cultured OBs for SCF mRNA expression at day 7 in Lnk KO and WT mice. Mouse heart and bone are used as positive controls for mouse endothelial and bone-related gene detection (not depicted) *, P < 0.05. Data averaged with SEM from three independent experiments. Experiments were obtained from triplicated assays. (c) The mineralized matrix formation was assessed by alizarin red staining 21 d after osteogenic culture in Lnk KO OBs with sKit (10 µg/ml) and in WT OBs in the presence of SCF (100ng/ml) with or without sKit (10 µg/ml). Upper panels show macroscopic images of whole culture dishes and lower panels show magnified images of nodule with mineralized matrix formation. Calcium content was measured by ELISA ( n = 5 each; d) in culture medium and BMP2 mRNA expression (e) was analyzed by real-time RT-PCR ( n = 3 each) in Lnk KO OBs with sKit (10 µg/ml) and WT OBs in the presence of SCF (100ng/ml) with or without sKit (10 µg/ml). *, P < 0.05 and **, P < 0.01. Experiments were obtained from triplicated assays.

Article Snippet: Purified recombinant human SCF and sKit were purchased from R&D Systems and supplemented concentrations of cytokines used for culture were as follows: SCF 100 ng/ml and sKit 10 μg/ml (Nakamura, 2004).

Techniques: Isolation, Cell Culture, Staining, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Overcoming CD226-related immune evasion in acute myeloid leukemia with CD38 CAR-engineered NK cells

doi: 10.1016/j.celrep.2024.115122

Figure Lengend Snippet:

Article Snippet: Recombinant human SCF protein , RnD Systems , 255-SC.

Techniques: Purification, Functional Assay, Virus, Recombinant, Binding Assay, Staining, Western Blot, Modification, Transfection, Fluorescence, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Cell Isolation, Plasmid Preparation, Generated, Sequencing, Software

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE 1. NS5A interacts specifically with DGAT1. A, shown is co-immuno- precipitation of endogenous DGAT1 with FLAG-tagged HCV proteins core, E1, NS2, NS3, NS4B, and NS5A, in Huh7 cells. After immunoprecipitation with -FLAG M2 affinity gel, the endogenous DGAT1 and FLAG-tagged proteins were detected by Western blots (WB) with respective antibodies. The input control was 12% of the whole-cell lysate used for each immunoprecipitation. NT, not tranfected. B, shown is co-IP of NS5A-GFP in Huh7 cells transfected with expression vectors for FLAG-DGAT1, FLAG-DGAT2, NS5A-GFP, and GFP proteins. The input control was 15% of the whole-cell lysate used for each immunoprecipitation. C, co-IP of NS5A with endogenous DGAT1 from HCV Jc1-infected Huh7.5 is shown. Normal rabbit IgG (Invitrogen) was used as a negative control (). The input control was 4% of the whole-cell lysate used for each immunoprecipitation.

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Immunoprecipitation, Western Blot, Control, Co-Immunoprecipitation Assay, Transfection, Expressing, Infection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE 2. DGAT1 is required for interaction of NS5A and core and forms a tripartite complex with these viral proteins. A, shown are sequential co-IP experiments of DGAT1, core, and NS5A. Top panel, 293T cells were transfected with plasmids expressing NS5A-GFP, FLAG-DGAT1, and HA-core. After 24 h, cells were lysed and subjected to Western blotting with -GFP, -core, and -FLAG antibodies. Middle panel, immunoprecipitation was performed with -HA antibody-conjugated agarose and subjected to Western blotting. Bottom panel, tandem immunoprecipitations were performed with -FLAG M2 affinity gel and -HA antibody-conjugated agarose. -FLAG M2 affinity gel was eluted with FLAG peptide, and the eluates were incubated with -HA antibody- conjugated agarose. Bound proteins were subjected to Western blotting. The input control was 12% of the whole-cell lysate used for the single immunopre- cipitation and 6% of that used for the tandem immunoprecipitation. B, immunoprecipitation of HA core in shRNA-expressing Huh7 cells transfected with NS5A-GFP and HA-core is shown. Cell lysates and bound proteins were analyzed by Western blot with -GFP, -DGAT1, and -core antibodies. NT, not transduced. The input control is 6% of the whole-cell lysate used for each immunoprecipitation. C, shown is real-time RT-PCR analysis of DGAT1 or DGAT2 mRNA expression levels in Huh7 cells transduced with the corresponding shRNAs. Data are the mean S.E.; n 3 independent experiments.

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Incubation, Control, shRNA, Quantitative RT-PCR, Transduction

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE 3. Intracellular localization of HCV proteins expressed individually and validation of the DGAT1 inhibitors. A, shown are representative images of Huh7-Lunet cells transfected with individual FLAG-tagged HCV proteins (FLAG-core, E1-FLAG, E2-FLAG, p7-FLAG, NS2-FLAG, NS4A-FLAG, NS4B-FLAG, and NS5A-FLAG). Cells were fixed and stained using -FLAG antibodies (green), LipidTox Red (LDs, red), and Hoechst (nucleus, blue) before imaging via epifluores- cence microscopy (scale bar 10 m). B, shown is quantification of triglycerides extracted from shRNA-transduced and inhibitor-treated Huh7-Lunet cells previously depleted of LDs. Lunet cells were transduced with shRNAs targeting DGAT1, DGAT2, or luciferase and then treated sequentially with Triacsin C for 18 h and DGAT1 inhibitors for 24 h. After depletion of intracellular LDs by triacsin C treatment, DGAT1 is the most potent enzyme in synthesizing triglyceride using exogenous fatty acid sources (11, 35). Significant inhibition of this DGAT1 activity was observed with concentrations of 20 and 75 M iA and iB, respectively. C, Huh7.5 cells transfected with HCV Luc-Jc1 viral RNA were treated with the DGAT1 inhibitors iA (20 M) or iB (75 M) or DMSO. After 48 h of treatment, viral particles secreted in the supernatants were used to infect naive Huh7.5 cells (Infected), and the producing cells were lysed (transfected). Shown are luciferase values expressed as percentage of DMSO control (mean S.E., n 3). NT, not treated. D, shown is quantification of triglycerides extracted from hepatoma cells treated with DMSO, 20 M iA, or 75 M iB for 48 h or 2.5 mg/ml of oleic acid-albumin for 16 h. In normal conditions, DGAT2 can compensate for the loss of DGAT1 activity, preventing any change in the triglyceride content of the cells.

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Biomarker Discovery, Transfection, Staining, Imaging, Microscopy, shRNA, Transduction, Luciferase, Inhibition, Activity Assay, Infection, Control

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE 4. NS5A localization at LDs is dependent on DGAT1 activity. Shown are representative images (scale bar 10 m) and quantification of Huh7-Lunet cells transfected with HCV Jc1 RNA (A and B), NS5A-GFP expres- sion vector (C and D), or NS5A-FLAG expression vector (E and F) and treated with DGAT1 inhibitors (iA at 20 M or iB at 75 M) or DMSO control for 48 h. Cells were fixed and stained with -NS5A or -FLAG antibodies (A and E, green), LipidTox Red (A and C) or far red (E) (LDs, red), and Hoechst (nucleus, blue). Green fluorescence in C arises directly from NS5A-GFP signal. Cells were analyzed by epifluorescence microscopy in A–D and by confocal microscopy in E–F. Colocalization of NS5A and LDs per cell was quantified by the auto- matic measurement program of the Zeiss axiovision software (mean of 40 cells S.E.) (B, D, and F).

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Control, Staining, Fluorescence, Epifluorescence Microscopy, Confocal Microscopy, Software

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE 5. NS5A association with LDs is dependent on DGAT1 activity. A, shown are Western blots of cell extracts (left panels) or isolated LD fractions (right panels) from Huh7-Lunet cells transfected with NS5A-GFP and incu- bated with oleate, DMSO, 20 M DGAT1 inhibitor iA described in Ref. (16), 75 M DGAT1 inhibitor iB (TOCRIS Bioscience). NT, not transfected. B, shown are Western blots analysis of cell extracts (left panels) or isolated LD fractions (right panels) from shRNA-expressing Huh7-Lunet cells. C, shown is real-time RT-PCR analysis of DGAT1 or DGAT2 mRNA expression levels in Huh7-Lunet cells transduced with the corresponding shRNAs (mean S.E.; n 3 inde- pendent experiments).

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Activity Assay, Western Blot, Isolation, Transfection, shRNA, Expressing, Quantitative RT-PCR, Transduction

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE 6. Overexpression of DGAT1 catalytically inactive mutant suppresses NS5A trafficking to LDs. A, shown is co-IP of NS5A-GFP with FLAG-DGAT1, FLAG-DGAT1-H426A, or FLAG-DGAT2 in Huh7 cells (Ø, empty vector). The input control is 15% of the whole-cell lysate used for each immunoprecipitation. B, shown are Western blots of the LD fraction from NS5A-GFP-transfected Huh7-Lunet cells expressing FLAG-DGAT1, FLAG-DGAT1-H426A, or FLAG-DGAT2. C, shown are Western blots of the LD fraction purified from FLAG-core-transfected Huh7-Lunet cells expressing FLAG-DGAT1, FLAG-DGAT1-H426A, or FLAG- DGAT2. D and E, Huh7-Lunet cells were cotransfected with an expression vector for FLAG-DGAT1 wild type or FLAG-DGAT1-H426A, and NS5A-GFP or HA core. Cells were fixed and stained using -FLAG and -core antibodies, LipidTox Red (LDs) and Hoechst (nucleus) before epifluorescence microscopy (scale bar 10 m).

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Over Expression, Mutagenesis, Co-Immunoprecipitation Assay, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Transfection, Expressing, Purification, Staining, Epifluorescence Microscopy

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE7.OverexpressionofDGAT1catalyticallyinactivemutantinhibits viral replication. A, shown are infectious titers released from Huh7.5 cells transfected with HCV Luc-Jc1 viral RNA and vectors expressing wild-type FLAG-DGAT1 or mutant FLAG-DGAT1-H426A or with an empty control vec- tor. Naive Huh7.5 cells were infected with cell supernatants of transfected cells and lysed 48 h after infection to analyze luciferase activity. The relative light units (RLU) are expressed as percentage relative to the empty vector- transfected control; mean S.E.; n 4; *, p 0.0002). B, shown are Western blots of cell extracts, lysed on day 6 after HCV RNA transfection.

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Transfection, Expressing, Mutagenesis, Control, Infection, Luciferase, Activity Assay, Plasmid Preparation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

doi: 10.1074/jbc.m112.434910

Figure Lengend Snippet: FIGURE 8. Model of HCV NS5A and core recruitment to DGAT1-generated lipid droplets. DGAT1 interacts with core and NS5A proteins and co-loads them onto DGAT1-generated LDs. This process is crucial for assembly of HCV virions. When NS5A access to LDs is dominantly suppressed by overexpress- ing a catalytically inactive mutant of DGAT1, infectious particles cannot form despite the presence of core around LDs (right panel).

Article Snippet: Antibodies and Reagents—The following antibodies were used: -core (clone C7–50, Affinity BioReagents), -NS5A (HCM-131–5, Austral Biologicals), -DGAT1 (H-255, Santa Cruz), -ADRP (H-80, Santa Cruz), -calreticulin (SPA-600, Stressgen), -tubulin (T6074, Sigma), -GFP (Santa Cruz), -FLAGM2 (Sigma), -FLAG (F7425, Sigma), -mouse Alexa 488 (Invitrogen), -rabbit Alexa 488 (Invitrogen), -mouse Ig HRP (eBioscience), -rabbit Ig HRP (eBioscience).

Techniques: Generated, Mutagenesis

Journal: Endocrinology

Article Title: Suppression of growth hormone (GH) Janus tyrosine kinase 2/signal transducer and activator of transcription 5 signaling pathway in transgenic mice overexpressing bovine GH.

doi: 10.1210/en.2003-1498

Figure Lengend Snippet: FIG. 1. Hepatic GHR, JAK2, STAT5a, and STAT5b abundance in liver from normal (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated ()], killed after 7.5 min, and livers were removed. Extracts were prepared, and equal amounts of solubilized liver protein were immunoprecipitated (IP) with GHR, JAK2, STAT5a, or STAT5b antibodies, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using the same IP antibodies. Quantification of protein abundance in liver was performed by scanning densitometry and expressed as a percentage of values measured in nonstimulated normal mice. Data are the mean SD of the indicated number of subsets (n) of different individuals, run in separate experiments: GHR and JAK2, n 4; STAT5a, n 7; STAT5b, n 6. *, P 0.05 vs. normal control mice.

Article Snippet: Antibodies STAT5a (L-20, catalog no. sc-1081), STAT5b (C-17, no. sc-835), CIS (N-19, no. sc-1529), PY (PY-99, no. sc-7020), SOCS-1 (H-93, no. sc-9021), SOCS-2 (H-74, no. sc-9022), and SOCS-3 (H-103, no. sc-9023) were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA); JAK2 (no. 06–255) and SIRP 1 (no. 06–729) antibodies were from Upstate Laboratories (Lake Placid, NY); SHP-2 (no. 610621) was from Transduction Laboratories (Lexington, KY).

Techniques: Transgenic Assay, Injection, Saline, Immunoprecipitation, SDS Page, Western Blot, Quantitative Proteomics, Control

Journal: Endocrinology

Article Title: Suppression of growth hormone (GH) Janus tyrosine kinase 2/signal transducer and activator of transcription 5 signaling pathway in transgenic mice overexpressing bovine GH.

doi: 10.1210/en.2003-1498

Figure Lengend Snippet: FIG. 2. Hepatic JAK2, STAT5a, and STAT5b tyrosine phosphorylation in normal mice (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated ()], and after 7.5 min, livers were excised. Extracts were prepared, and equal amounts of solubilized liver protein were immunoprecipitated (IP) with JAK2, STAT5a, or STAT5b antibodies, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using PY. Protein phosphorylation was quantified by scanning densitometry and expressed as a percentage of the corresponding values in GH-stimulated normal mice. Data are the mean SD of four subsets of different individuals run in separate experiments (n 4). *, P 0.001 vs. saline-treated normal mice.

Article Snippet: Antibodies STAT5a (L-20, catalog no. sc-1081), STAT5b (C-17, no. sc-835), CIS (N-19, no. sc-1529), PY (PY-99, no. sc-7020), SOCS-1 (H-93, no. sc-9021), SOCS-2 (H-74, no. sc-9022), and SOCS-3 (H-103, no. sc-9023) were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA); JAK2 (no. 06–255) and SIRP 1 (no. 06–729) antibodies were from Upstate Laboratories (Lake Placid, NY); SHP-2 (no. 610621) was from Transduction Laboratories (Lexington, KY).

Techniques: Phospho-proteomics, Transgenic Assay, Injection, Saline, Immunoprecipitation, SDS Page, Western Blot

Journal: Endocrinology

Article Title: Suppression of growth hormone (GH) Janus tyrosine kinase 2/signal transducer and activator of transcription 5 signaling pathway in transgenic mice overexpressing bovine GH.

doi: 10.1210/en.2003-1498

Figure Lengend Snippet: FIG. 4. Membrane-associated GHR, JAK2, STAT5a, and STAT5b abundance in liver microsomes from normal (N) and PEPCK-bGH transgenic (T) mice. Animals were injected ip with normal saline [nonstimulated (–)] or oGH (5 mg/kg) [GH-stimulated ()]. Mice were killed after 7.5 min, and livers were removed. Microsomes were prepared, separated by SDS-PAGE, and subjected to immunoblot analysis (WB) using GHR, JAK2, STAT5a, or STAT5b antibodies. Quantification of protein abundance in liver microsomes was performed by scanning densitometry and expressed as a percentage of the mean value measured in nonstimulated normal mice. Data are the mean SD of four subsets of different individuals run in two separate experiments (n 4). †, P 0.05 vs. normal control mice; #, P 0.01 vs. nonstimulated normal mice; *, P 0.001 vs. normal control mice.

Article Snippet: Antibodies STAT5a (L-20, catalog no. sc-1081), STAT5b (C-17, no. sc-835), CIS (N-19, no. sc-1529), PY (PY-99, no. sc-7020), SOCS-1 (H-93, no. sc-9021), SOCS-2 (H-74, no. sc-9022), and SOCS-3 (H-103, no. sc-9023) were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA); JAK2 (no. 06–255) and SIRP 1 (no. 06–729) antibodies were from Upstate Laboratories (Lake Placid, NY); SHP-2 (no. 610621) was from Transduction Laboratories (Lexington, KY).

Techniques: Membrane, Transgenic Assay, Injection, Saline, SDS Page, Western Blot, Quantitative Proteomics, Control