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: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: Generation of Col15a1fl/fl mice and smooth muscle cell (SMC)-specific Col15a1fl/fl SMC lineage tracing mice. A: schematic of collagen type XV α1-gene (Col15a1) targeting vector injected into embryonic stems cells. Vector insertion resulted in selective neomycin resistance of embryonic stem cells. B: schematic of Col15a1 genome targeted insertion contained within chimera pups. C: schematic showing cross of Col15a1 chimera with Flpe recombinase mice results in excision of neomycin selectivity. This represents the Col15a1 locus in the animals used in our studies. D: schematic representation of SMC-specific Col15a1fl/fl SMC lineage tracing mice before and after tamoxifen treatment. Myh11P is the Myh11 promoter. Cre-ERT2 is the estrogen receptor bound Cre recombinase.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Plasmid Preparation, Injection

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: Genotyping and excision primers for Col15a1

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: Validation of specificity of SMC Col15a1 knockout, SMC lineage tracing mice. A: excision of Col15a1 occurs only in presence of tamoxifen and shows nearly complete recombination in SMCs. B: SMC Col15a1 knockout is highly specific to SMC-rich vessels (aorta and carotid). C and D: staining for COL15A1 in advanced lesions from 18 wk Western diet-fed SMC lineage tracing [yellow fluorescent protein (YFP)]+/+ Col15a1wt/wt, apoliprotein E (Apoe)−/− (n = 11) and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 13) mice shows a significant reduction of COL15A1 in both the media and lesion of SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− mice. Values represent means ± SE. Scale bars = 100 µm. ‘P value was determined by an unpaired, two-tailed t-test with Welch’s correction; ^P value was determined by Mann-Whitney U-test.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Biomarker Discovery, Knock-Out, Staining, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: Loss of SMC-derived COL15A1 resulted in a marked decrease in lesion size. A: representative images of brachiocephalic artery (BCA) cross sections from SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 11) and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 13) mice fed a Western diet for 18 wk stained with Movat reagent for lesion morphometry. Scale bars = 100 µm. B: quantification of lesion area, external elastic lamina (EEL) area, media area, and lumen area measurements from Movat staining of BCAs. P values were determined by two-way ANOVA (repeated measures). C and D: representative images (C) and quantification (D) of Sudan IV-stained aortas from SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 9) and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 6) mice. Values represent means ± SE. ^P value was determined by Mann-Whitney U-test.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Derivative Assay, Western Blot, Staining, MANN-WHITNEY

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: No differences in cholesterol, triglycerides, or body weight between SMC YFP +/+ Col15a1 wt/wt , Apoe −/− and SMC YFP +/+ Col15a1 Δ/Δ , Apoe −/− mice

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: Genetic inactivation of Col15a1 in SMCs reduced collagen fiber content and maturation as well as vessel elasticity. A: representative images of picrosirius red-stained BCAs under polarized light of SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 11) and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 13) mice. Scale bars = 100 µm. Quantification of total collagen content per lesion (B) and media (C) area. D: representative images of collagen maturation by LC-Polscope analysis where pixel intensity is proportional to birefringent, organized fibrillar collagen. Average retardance measurements indicated a decrease in lesion (E) and media (F) collagen organization with SMC Col15a1 knockout. Heat map represents the amplitude of retardance (nm) from 0 (black) to 39.5 (red). Scale bars = 100 µM. P value was determined by an unpaired, two-tailed t-test; ‘P value was determined by an unpaired, two-tailed t-test with Welch’s correction; ^P value was determined by a Mann-Whitney U-test. G and H: quantification of pressure myography to assess active and passive tone of carotid arteries of 18-wk Western diet-fed SMC Col15a1wt/wt, Apoe−/− (n = 6) and SMC Col15a1Δ/Δ, Apoe−/− (n = 5) mice. G: no significant differences were detected at any point in active tone. H: passive tone was significantly increased in SMC Col15a1Δ/Δ, Apoe−/− compared with SMC Col15a1wt/wt, Apoe−/− animals. Values represent means ± SE. For passive tone, P values were determined by an unpaired, two-tailed t-test at each pressure. *P < 0.05; +P < 0.054.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Staining, Knock-Out, Two Tailed Test, MANN-WHITNEY, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: Loss of SMC-specific COL15A1 resulted in overall reductions in the number of YFP+ SMC-derived cells within the lesions but an increase in YFP−galectin 3 (LGALS3)+ macrophage cells. A: representative immunofluorescence images of BCA sections of SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 11) and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 13) mice showing a marked decrease in overall cell number, a decrease in YFP+/DAPI+ cells, and an increase in YFP−LGALS3+/DAPI+ cells within lesions of SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− mice. Scale bars = 100 µm. B: quantification of cell number per lesion stained positive for DAPI, YFP, LGALS3, or smooth muscle α-actin (ACTA2) averaged across four locations of the BCA. C: quantification of percentage of SMCs (YFP) and non-SMC-derived macrophages (YFP−LGALS3+) over total number of cells (DAPI+) within lesions averaged across four separate locations of the BCA. D: quantification of cell density of the lesion by examining cell number normalized to lesion area (μm2). Given that SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− have more acellular, necrotic core regions in their lesions as compared with that of SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− mice, we also quantified cell density of the lesion (E) by examining cell number normalized to the lesion area (μm2) with the necrotic core regions excluded. Values represent means ± SE. P values were determined by an unpaired, two tailed t-test. ^P value was determined by a Mann-Whitney U-test.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Derivative Assay, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: SMC-derived cell populations were reduced with SMC Col15a1 knockout. A: no changes were observed in the percentage of ACTA2+/DAPI+ cells but a significant increase was found in the percentage of LGALS3+/DAPI+ cells in SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 13) compared with SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 11) lesions. B: a significant decrease in SMC-derived ACTA2+ (YFP+ACTA2+/DAPI+) cells and a near significant increase in non-SMC-derived ACTA2+ (YFP−ACTA2+/DAPI+) cell populations were also found in SMC Col15a1 knockout compared with wild type. C: no significant differences were seen in the proportion of SMCs able to express ACTA2 (YFP+ACTA2+/YFP+) or LGALS3 (YFP+LGALS3+/YFP+) as a consequence of SMC Col15a1 knockout. Values represent the average of three locations across the BCA for each genotype. Values represent means ± SE. ‘P value was determined by an unpaired two-tailed t-test analysis with Welch’s correction.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Derivative Assay, Knock-Out, Two Tailed Test

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: SMC-specific Col15a1 knockout was associated with a decrease in lesion SMC proliferation. A: representative immunofluorescence images of BCA sections stained for proliferating cells (MKi67+) within SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 11) and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 13) lesions. B: higher magnification and single marker immunostaining of the white box region in A. Arrows indicate subpopulations of proliferating cells including SMC (YFP+MKi67+, yellow arrows), non-SMC-derived macrophages (YFP−LGALS3+MKi67+, white arrows), and SMC-derived macrophage-like cells (YFP+LGALS3+MKi67+, red arrows). Scale bars = 100 µm (A) and 25 µm (B). B: quantification of the percentage of proliferating cells within the lesion (MKi67+/DAPI+) showed no difference between SMC YFP+/+ Col15a1wt/wt, Apoe−/− and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− mice. C: however, there was a significant decrease in the percentage of SMC-derived cells (YFP+MiKi67+/DAPI+) and an increase in the percentage of non-SMC-derived macrophages (YFP−MKi67+LGALS3+/DAPI+) proliferating within SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− compared with SMC YFP+/+ Col15a1wt/wt, Apoe−/− lesions. Values represent means ± SE of one location of the BCA. ^P value was determined by Mann-Whitney U-test analysis. P value was determined by an unpaired two-tailed t-test.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Knock-Out, Immunofluorescence, Staining, Marker, Immunostaining, Derivative Assay, MANN-WHITNEY, Two Tailed Test

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: SMC Col15a1 knockout resulted in a decrease in lesion cell death. A: representative immunofluorescent images of SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 11) and SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 13) lesions stained for the cell death marker cleaved caspase-3 (CASP3+). B and C: quantification of CASP3+ cells counted per lesion (B) and percentages of CASP3+ cells within lesions (C; CASP3+/DAPI+) showed that SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− mice have significantly fewer dying cells compared with SMC YFP+/+ Col15a1wt/wt, Apoe−/− mice. Values represent means ± SE. Scale bars = 100 µm and on zoom in images 25 µm. ^P value from a Mann-Whitney U-test.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Knock-Out, Staining, Marker, MANN-WHITNEY

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: SMC-specific Col15a1 knockout resulted in downregulation of immune cell processes that impact atherosclerotic plaque formation. RNA-seq analysis of BCA, aortic arch, and carotid artery specimens from 18-wk Western diet-fed SMC YFP+/+ Col15a1Δ/Δ, Apoe−/− (n = 3) compared with SMC YFP+/+ Col15a1wt/wt, Apoe−/− (n = 3) mice. A: Ingenuity Pathway Analysis showed dysregulation of inflammatory pathways. The red line marks the cut off for significantly enriched (adjusted P ≤ 0.05) pathways. Enrichment is shown as –log10 of the adjusted P value. B: Upstream Regulator Analysis was performed on the top ~3,000 differentially expressed genes containing a log fold change of ±0.30 from the RNA-seq to identify master regulators that are either inhibited or activated as a consequence of SMC Col15a1 knockout.

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Knock-Out, RNA Sequencing, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

doi: 10.1152/ajpheart.00029.2017

Figure Lengend Snippet: COL15A1 is pervasive within advanced human atherosclerotic plaques and localized to the fibrous cap and intraplaque microvessels. A representative image of a human coronary artery lesion from 1 of 6 patients analyzed who underwent coronary artery bypass surgery is shown. Serial sections were stained with COL15A1 (A) or traditional macrophage (CD68; B) and SMC marker (ACTA2) genes. C and D: boxes (1 and 2) represent regions of higher magnification to highlight COL15A1 presence in the ACTA2-rich fibrous cap (C and D) and intraplaque microvessels (D, blue arrows) with little to no COL15A1 staining in CD68-rich regions (D).

Article Snippet: Col15a1 excision was assessed by Taqman Copy Number Variant Assay using a probe targeting exon 1 of Col15a1 (Mm00558775_cn, Life Technology; ).

Techniques: Staining, Marker

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: Next, the membranes were incubated with primary antibodies targeting pro-Collagen type 1A1, SMAD4, LaminB, and β-actin (Santa Cruz, sc-293182, sc-7966, sc6216, and sc4778, respectively); α-SMA (Abcam, ab7817); Nav1.5 (Almone Labs, AC-005); Fibronectin (Millipore, MAB1940); TGF-beta receptor I (Sigma, SAB1300113); TGF-β1, TGF-beta receptor II, phospho-SMAD2/3 (Cell signaling, 3711, 11888, and 8828, respectively); as well as GAPDH (Mbl, M171).

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: Next, the membranes were incubated with primary antibodies targeting pro-Collagen type 1A1, SMAD4, LaminB, and β-actin (Santa Cruz, sc-293182, sc-7966, sc6216, and sc4778, respectively); α-SMA (Abcam, ab7817); Nav1.5 (Almone Labs, AC-005); Fibronectin (Millipore, MAB1940); TGF-beta receptor I (Sigma, SAB1300113); TGF-β1, TGF-beta receptor II, phospho-SMAD2/3 (Cell signaling, 3711, 11888, and 8828, respectively); as well as GAPDH (Mbl, M171).

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: Next, the membranes were incubated with primary antibodies targeting pro-Collagen type 1A1, SMAD4, LaminB, and β-actin (Santa Cruz, sc-293182, sc-7966, sc6216, and sc4778, respectively); α-SMA (Abcam, ab7817); Nav1.5 (Almone Labs, AC-005); Fibronectin (Millipore, MAB1940); TGF-beta receptor I (Sigma, SAB1300113); TGF-β1, TGF-beta receptor II, phospho-SMAD2/3 (Cell signaling, 3711, 11888, and 8828, respectively); as well as GAPDH (Mbl, M171).

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: Next, the membranes were incubated with primary antibodies targeting pro-Collagen type 1A1, SMAD4, LaminB, and β-actin (Santa Cruz, sc-293182, sc-7966, sc6216, and sc4778, respectively); α-SMA (Abcam, ab7817); Nav1.5 (Almone Labs, AC-005); Fibronectin (Millipore, MAB1940); TGF-beta receptor I (Sigma, SAB1300113); TGF-β1, TGF-beta receptor II, phospho-SMAD2/3 (Cell signaling, 3711, 11888, and 8828, respectively); as well as GAPDH (Mbl, M171).

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: Next, the membranes were incubated with primary antibodies targeting pro-Collagen type 1A1, SMAD4, LaminB, and β-actin (Santa Cruz, sc-293182, sc-7966, sc6216, and sc4778, respectively); α-SMA (Abcam, ab7817); Nav1.5 (Almone Labs, AC-005); Fibronectin (Millipore, MAB1940); TGF-beta receptor I (Sigma, SAB1300113); TGF-β1, TGF-beta receptor II, phospho-SMAD2/3 (Cell signaling, 3711, 11888, and 8828, respectively); as well as GAPDH (Mbl, M171).

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: 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: eLife

Article Title: Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure

doi: 10.7554/eLife.37131

Figure Lengend Snippet:

Article Snippet: Whole-mount fluorescent immunostains were performed using standard protocols and a mouse monoclonal antibody against ZO-1 (ZO1-1A12, Thermo Fisher Scientific, Waltham, MA), a rabbit polyclonal antibody against collagen II (ab209865, Abcam), and a rabbit polyclonal antibody against laminin (L9393, Sigma).

Techniques: Mutagenesis, Plasmid Preparation, Recombinant, Sequencing, In Situ, Purification, Electron Microscopy, Software