Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 5. Effect of SER5 on HIV-1 particles pseudotyped with different envelopes (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with envelopes of HIV-1 BaL (red), FeLV-A (green), FeLV-B (blue) or amphotropic (ampho) Env (orange) were produced in the presence of increasing amounts of feline SERINC5 (feSER5) or (B) human SERINC5 (huSER5) (0, 100, 200 or 400 ng). Viral infectivity was determined after normalization for reverse transcriptase activity by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001 to 0.01 very significant (**), 0.01 to 0.05 significant (*), >0.05 not significant (ns). (C) SERINC5 stability in the presence of increasing amounts of FeLV-B envelope. HEK293T cells were transfected with fe/huSER5 and increasing amounts of FeLV-B envelope expression plasmids or empty vector as control. Cells were harvested and analyzed by immunoblotting using anti-p85/70, anti-HA and anti-tubulin antibodies. a p85/70 detects envelope (SU-TM p85) and SU only (p70), a HA detects HA-SER5.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Infection, Reverse Transcription, Activity Assay, Luciferase, Transfection, Expressing, Control, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 6. Generation of FeLV glycoGag expression constructs. (A) Sequences of FeLV-A and FeLV-B glycoGags. The highlighted amino acids correspond to disagreements, which are colored according to their side chain chemistry. Cytoplasmic and transmembrane domains are indicated. (B) Plasmids expressing the HA-tagged FeLV glycoGag were produced by amplifying the glycoGag from pFGA-5 and pFGB (FeLV-A and FeLV-B glycoGag respectively), yielding a 9.6 kDa protein. (C) HEK293T cells were transfected with the generated expression plasmids, harvested two days post-transfection and analyzed by immunoblotting using anti-HA and anti-tubulin antibodies.

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Expressing, Construct, Produced, Transfection, Generated, Western Blot

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 7. Effect of FeLV glycoGag on HIV-1 in the presence of SER5. (A) HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 BaL envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) together with FeLV-A glycoGag, FeLV-B glycoGag or empty vector. Particles were normalized by RT activity and used for infections. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns). (B) Effect of SERINC and glycoGag on HIV-1 cell-to-cell transmission. HIV-1 reporter vectors (3-plasmid system, no Nef) pseudotyped with HIV-1 envelope BaL were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV-A or -B glycoGag. After transfection, virus producer HEK293T cells were co-cultured with HEK293T transfected with expression plasmids for human CD4 and human CCR5. The infectivity determined by quantification of luciferase activity of a vector that is detectable only after reverse transcription of the spliced luciferase gene. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001– 0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase, Transmission Assay, Transfection, Virus, Cell Culture, Expressing, Reverse Transcription

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 8. Effect of FeLV glycoGag or FeLV-B envelope on FIV in the presence of SER5. FIV particles (3- plasmid system) pseudotyped with FIV EE14 envelope were produced in the absence or presence of feline SERINC5 (feSER5) or human SERINC5 (huSER5) and FeLV glycoGag or in the absence of FIV EE14 envelope and glycoGag and pseudotyped by FeLV-B envelope. Particles were normalized by RT activity and used for infection. The infectivity determined by quantification of luciferase activity. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01–0.05 significant (*), >0.05 not significant (ns).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Plasmid Preparation, Produced, Activity Assay, Infection, Luciferase

Journal: Journal of molecular biology

Article Title: Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5.

doi: 10.1016/j.jmb.2021.167421

Figure Lengend Snippet: Figure 9. Comparison of delta Ct values of feSER5 in transfected HEK293T cells and in cat cells and surface expression of feSER5 in the presence of different FeLV proteins. (A) HEK293T cells were transfected with different amounts of feSER5 plasmid. The endogenous feSER5 were evaluated in feline CRFK cell lines and PBMC or macrophages from one cat. (B) HEK293T cells were cotransfected with feSER5-iHA and empty plasmid, Nef, MLV glycoGag , FeLV-A/B glycoGag or FeLV-A/B envelope. The percent of feSER5 positive cells was evaluated by flow cytometry analysis and the total of feSER5 positive cells were scaled to 100%. Shown are means plus standard deviations (error bars) of a representative experiment performed three times in triplicate. Asterisks represent statistically significant differences: P value <0.001 extremely significant (***), 0.001–0.01 very significant (**), 0.01– 0.05 significant (*), >0.05 not significant (ns). (C) Representative plots of one of three experiments showing the surface expression of feSER5-iHA (HA-Alexa Fluor 488). Green fluorescence was quantified using the FITC-A channel vs. FSC-A (10,000 cells were counted).

Article Snippet: The glycoGag expression in the FeLV plasmids was evaluated by using anti-FeLV p27 antibody (1:7500 dilution; PF12J-10A: sc-65623; Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg.

Techniques: Comparison, Transfection, Expressing, Plasmid Preparation, Cytometry

Journal: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet: SCN5A knockdown promotes the expression of fibrogenic signaling (A) Upper panel: Knockdown of SCN5A gene in human cardiac fibroblasts by targeting SCN5A gene using shRNA lentivirus. Lower panel : fibroblast grown after SCN5A gene knockdown and morphology analyzed microscopically, Scale bar 350 μm (representative pictures shown). (B) The protein expression of Nav1.5 after the knockdown of the SCN5A gene in HCF represents a 50% decrease in Nav1.5 protein expression. Data are expressed as mean ± SEM, paired t-test, ∗∗ p < 0.01, n = 5 independent experiments. (C) Representatives immunoblot and quantitative analysis showing the expression of pro-Col1agen 1A1, α-SMA, and fibronectin in SCN5A knockdown and control HCF normalized with the internal control group. Data are expressed as mean ± SEM, paired t-test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n = 6 independent experiments. (D) There were higher soluble collagen-type 1 levels measured in a conditioned medium (serum-free) of SCN5A knockdown HCF than that from control cells Data are expressed as mean ± SEM, paired t-test, ∗∗∗ p < 0.001, n = 6 independent experiments. SCN5A shRNA: SCN5A knockdown HCF.

Article Snippet: Anti-Pro-Collagen type 1A1 , Santa Cruz , Cat# sc-293182; RRID: AB_2797597.

Techniques: Knockdown, Expressing, shRNA, Western Blot, Control

Journal: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet: MiR-452-5p mimic restored the fibrogenic phenotype in SCN5A knockdown HCF (A) Immunoblot and quantitative analysis of the expression in protein levels of pro-Collagen type 1A1 and fibronectin in control, and SCN5A knockdown HCF, miR-NTC, and miR-452-5p-mimic groups. Data are expressed as mean ± SEM, paired t-test, ∗∗ p < 0.01, n = 6 independent experiments). (B) Soluble collagen-type1 level measured in conditioned medium from SCN5A knockdown HCF as compared to control and SCN5A knockdown HCF treated without or with miR-NTC and miR-452-5p mimic. Data are expressed as mean ± SEM, paired t-test, ∗ p < 0.05, ∗∗ p < 0.01, n = 5 independent experiments. (C) Immunohistochemistry shows increased expression of α-SMA in SCN5A knockdown HCF and this expression was significantly reduced upon the treatment of miR-452-5p mimic as compared to both control and miR-NTC groups. α-SMA (green), DAPI (blue). Scale bar: 90 μm. (D) Immunoblots and quantitative analysis of α-SMA in SCN5A knockdown HCF as compared to control and SCN5A knockdown HCF, miR-NTC, and miR-452-5p-mimic groups. Data are expressed as mean ± SEM, paired t-test, ∗∗ p < 0.01. ∗∗∗ p < 0.001, n = 6 independent experiments. (E) Representative images of migration at 0 h and 10 h post-wounding. Scale bar: 300 μm. Graph showing the cell migration distances (μm) of control, SCN5A knockdown HCF, miR-NTC, and miR-452-5p mimic treated groups. Data are expressed as mean ± SEM, paired t-test, ∗∗∗ p < 0.001, n = 10 independent experiments. SCN5A shRNA: SCN5A knockdown HCF. miR-NTC: miR negative control.

Article Snippet: Anti-Pro-Collagen type 1A1 , Santa Cruz , Cat# sc-293182; RRID: AB_2797597.

Techniques: Knockdown, Western Blot, Expressing, Control, Immunohistochemistry, Migration, shRNA, Negative Control

Journal: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet: Systemic delivery of AAV miR452 ameliorates fibrosis in isoproterenol-induced HF rats (A) Nav1.5 protein expression in left ventricular tissues of HF and control rats. Data are expressed as mean ± SEM, paired t-test, ∗∗ p < 0.01, n = 3 independent experiments. (B) MiR-452-5p mimic delivery through AAV9 increased the miR-452 expression in left ventricular tissues which was initially reduced after HF development as compared to control, measured via qRT-PCR. Data are expressed as mean ± SEM, one-way ANOVA followed by Tukey’s multiple comparison test, ∗∗∗ p < 0.001, n = 5–6 independent experiments. (C) Immunoblots and quantitative analysis of pro-collagen type 1a1, fibronectin, α-SMA in LV tissues of HF, and AAV miR452 compared with control. Data are expressed as mean ± SEM, one-way ANOVA followed by Tukey’s multiple comparison test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n = 5–6 independent experiments. (D) Immunoblots and quantitative analysis of TGF-β signaling including TGF-β1, TGF-βRI, RII, p -SMAD2/3, SMAD4 in LV tissues of HF (induced by subcutaneous injection of isoproterenol 100 mg/kg once a week for 2 weeks) and HF rats treated with AAV miR452 (3.0 x10 10 genome copies per rat via tail vein, once a week for 2 weeks) compared with control (received normal saline). Data are represented as mean ± SEM, one-way ANOVA followed by Tukey’s multiple comparison test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n = 5–6 independent experiments.

Article Snippet: Anti-Pro-Collagen type 1A1 , Santa Cruz , Cat# sc-293182; RRID: AB_2797597.

Techniques: Expressing, Control, Quantitative RT-PCR, Comparison, Western Blot, Injection, Saline

Journal: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet: The proposed mechanism of shielding potential of miR-452-5p in cardiac fibroblasts against SCN5A knockdown escalated cardiac fibrogenesis The expression level of miR-452-5p in normal HCF serves to restrain the activity of SMAD4 protein by binding to the 3ˊUTR of SMAD4 mRNA, therefore limiting its activity. However, in the case of SCN5A knockdown, the quantity of miR-452-5p is considerably reduced, impairing its capacity to bind with 3′UTR of SMAD4 mRNA and thereby increasing SMAD4 activity. The increase in SMAD4 activity causes the TGF-β to be overexpressed which activates the canonical TGF-β signaling pathway by increasing the phosphorylation of downstream signal transducers SMAD2 and SMAD3. Following phosphorylation, these form a heterodimer with SMAD4 and translocate into the nucleus, where they increase the expression of fibrogenesis-related genes such as pro-Collagen 1A1, fibronectin, and fibroblasts differentiation by overexpressing α-SMA, and increased cell migration leading to cumulative effect on fibrogenesis in SCN5A knockdown condition.

Article Snippet: Anti-Pro-Collagen type 1A1 , Santa Cruz , Cat# sc-293182; RRID: AB_2797597.

Techniques: Knockdown, Expressing, Activity Assay, Protein Binding, Phospho-proteomics, Migration

Journal: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet:

Article Snippet: Anti-Pro-Collagen type 1A1 , Santa Cruz , Cat# sc-293182; RRID: AB_2797597.

Techniques: Virus, Recombinant, Transfection, Membrane, Western Blot, Protein Extraction, Reverse Transcription, Qubit Protein Assay, Collagen Assay, Enzyme-linked Immunosorbent Assay, Extraction, Plasmid Preparation, Software, RNA Sequencing

Journal: PLoS ONE

Article Title: Ovarian stromal cells as a source of cancer-associated fibroblasts in human epithelial ovarian cancer: A histopathological study

doi: 10.1371/journal.pone.0205494

Figure Lengend Snippet: Antibody and staining conditions.

Article Snippet: Collagen type4 , mouse , CIV22 , Cell Marque, Rocklin, CA , 1:500 , Citrate pH6 , ImmPRESS-AP , Vector Red (red) .

Techniques: Staining, Plasmid Preparation