Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 2. Effect of the TRPC5 activator AM237 on acetylcholine (ACh)-induced relaxation and contraction in the normal-fat diet (NFD) mouse aorta. (a) TRPC5 protein expression in mouse aortic endothelial cells (MAoECs) from NFD ( n = 10) and high-fat diet (HFD)-induced obese ( n = 11) mice. (b) Co-immunostaining of TRPC5 and CD31 in mouse aortic rings (scale bar, 50 𝜇m). (c–e) Representative traces (c) and summary data (d, e) of phenylephrine (Phe)-precontracted ACh-induced relaxation followed by contraction at high concentrations in NFD ( n = 7) and AM237 (100 nmol/L)-pretreated NFD ( n = 6) aortas. (f–h) Original recordings (f) and data summary (g, h) showing the effect of AM237 (100 nmol/L) on ACh-induced contraction in the NFD aorta with or without endothelium ( + endo CTL, n = 5; + endo AM237, n = 5; –endo CTL, n = 4; –endo AM237, n = 4). Mean ± SEM; A, ∗ P < 0.05 vs NFD, Student’s unpaired two-tailed t test; d, ∗ P < 0.05, NS, no significant difference vs NFD, two-way ANOVA followed by Bonferroni test; e, ∗ P < 0.05, NS, no significant difference vs CTL, Student’s unpaired two-tailed t test; g, ∗ P < 0.05 vs + endo CTL, two-way ANOVA followed by Bonferroni test; h, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237, one-way ANOVA followed by Turkey’s multiple comparisons test.

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Expressing, Immunostaining, Two Tailed Test

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 3. Effect of TRPC5 inhibition on acetylcholine (ACh)-induced vaso- constriction in the high-fat diet (HFD)-induced obese mouse aorta. (a, b) Representative traces (a) and data summary (b) showing ACh-induced contrac- tion is attenuated by the TRPC5 inhibitor clemizole (20 𝜇mol/L), knockout of TRPC5, and the removal of endothelium (CTL, n = 5; clemizole, n = 6; TRPC5 − / − , n = 5; CTL(–Endo), n = 6; mean ± SEM; b, left, ∗ P < 0.05 vs CTL, two-way ANOVA followed by Bonferroni test; right, ∗ P < 0.05 vs CTL, one-way ANOVA followed by Dunnett’s multiple comparisons test).

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Inhibition, Knock-Out

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 4. TRPC5 regulates contractions via cytosolic phospholipase A 2 (cPLA 2 ) in the high-fat diet (HFD)-induced obese mouse aorta. (a) Western blots and analysis of cPLA 2 and phosphorylated cPLA 2 (p-cPLA 2 ) expression in normal-fat diet (NFD, n = 15), AM237 (100 nmol/L)-treated NFD ( n = 9), HFD-induced obese ( n = 14), clemizole (20 𝜇mol/L)-treated HFD ( n = 7), and TRPC5 − / − -HFD ( n = 5) mouse aortic endothelial cells (MAoECs). (b) Dose-dependent effect of AM237 on p-cPLA 2 levels in NFD MAoECs. AM237 (nmol/L), 0, n = 16; 50, n = 11; 100, n = 16; 200, n = 10. (c) Dose-dependent effect of clemizole on p-cPLA 2 levels in HFD MAoECs ( n = 5). (d) Representative fluorescence images of the Bis-BODIPY TM FL C 11 -PC stained en-face aorta and analysis of PLA 2 activity in endothelial cells of the NFD ( n = 9), AM237 (100 nmol/L)-pretreated NFD ( n = 7), HFD ( n = 17), clemizole (20 𝜇mol/L)-treated HFD ( n = 9), and TRPC5 − / − HFD ( n = 12) mouse aorta (scale bars, 10 𝜇m). (e) Acetylcholine (ACh)-induced contraction in HFD ( n = 4) and MAFP (10 𝜇mol/L)-treated HFD ( n = 6) mouse aortic rings. (f) ACh-induced contraction in the NFD ( n = 5), AM237 (100 nmol/L)-pretreated NFD ( n = 5), and AM237 (100 nmol/L) + MAFP (30 𝜇mol/L)-pretreated NFD ( n = 6) mouse aorta. Mean ± SEM; a, ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, NS, no significant difference, Kruskal-Wallis and Dunn’s post hoc non-parametric test (p-cPLA 2 ) and one-way ANOVA followed by Turkey’s multiple comparisons test (cPLA 2 ); b, ∗ P < 0.05, NS, no significant difference vs no AM237, one-way ANOVA followed by Dunnett’s multiple comparisons test; c, ∗ P < 0.05, NS, no significant difference vs no clemizole, one-way ANOVA followed by Dunnett’s multiple comparisons test; d, ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, one-way ANOVA followed by Turkey’s multiple comparisons test; e, ∗ P < 0.05 vs CTL, two-way ANOVA followed by Bonferroni test (left) and Student’s unpaired two-tailed t test (right); f, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Turkey’s multiple comparisons test (right).

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Western Blot, Expressing, Staining, Activity Assay, Two Tailed Test

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 6. Role of COX-2 in TRPC5-regulated vasoconstriction in the mouse aorta. (a) Western blots and analysis of COX-1 and COX-2 expression in normal-fat diet (NFD, n = 5), AM237 (100 nmol/L) pre-treated NFD ( n = 5), high-fat diet (HFD, n = 5), clemizole (20 𝜇mol/L) pre-treated HFD ( n = 5), and TRPC5 − / − HFD ( n = 5) mouse aortic endothelial cells (MAoECs). (b) Effect of the COX inhibitors NS-398 (3 𝜇mol/L) ( n = 5), VAS-2870 (30 𝜇mol/L) ( n = 3), and indomethacin (indo, 1 𝜇mol/L, n = 3) on acetylcholine (ACh)-induced contraction in the HFD mouse aorta (CTL, n = 3). (c) Effect of the COX-2 inhibitor NS-398 (3 𝜇mol/L) on ACh-induced contraction in the AM237 (100 nmol/L)-pretreated NFD mouse aorta (CTL, n = 5; AM237, n = 7; AM237 + NS-398, n = 7). Mean ± SEM; a, ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, NS, no significant difference, Kruskal-Wallis and Dunn’s post hoc non-parametric test (COX-1) and one-way ANOVA followed by Turkey’s multiple comparisons test (COX-2); b, ∗ P < 0.05, NS, no significant difference vs CTL, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Dunnett’s multiple comparisons test (right); c, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Turkey’s multiple comparisons test (right) .

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Western Blot, Expressing

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 5. TRPC5 contributes to acetylcholine (ACh)-induced Ca 2+ entry into endothelial cells of high-fat diet (HFD)-induced obese mouse aortas. (a, b) Representative traces (a) and data summary (b) showing an ACh (10 𝜇mol/L)- induced [Ca 2 + ] i rise in aortic endothelial cells from normal-fat diet (NFD) and HFD mice (NFD, n = 7; NFD-AM237 (100 nmol/L), n = 5; HFD, n = 5; HFD- clemizole (20 𝜇mol/L), n = 6; HFD-TRPC5 − / − , n = 6; mean ± SEM; ∗ P < 0.05 vs NFD, # P < 0.05 vs HFD, one-way ANOVA followed by Turkey’s multiple com- parisons test).

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques:

Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: Fig. 7. PGF 2 𝜶and PGE 2 are involved in TRPC5-related contraction in the high-fat diet (HFD)-induced obese mouse aorta. (a) Enzyme immunoassay (EIA) showing the PGF 2 𝛼( n = 7), PGE 2 ( n = 7), PGD 2 ( n = 5), PGI 2 ( n = 5), and 8-isoprostanes ( n = 6) levels in normal-fat diet (NFD) and HFD mouse aortic rings after exposure to acetylcholine (ACh, 10 𝜇mol/L). (b) Effect of clemizole (20 𝜇mol/L), MAFP (10 𝜇mol/L), NS-398 (3 𝜇mol/L), –endo, and knockout of TRPC5 − / − on ACh-induced PGF 2 𝛼and PGE 2 release in the ACh-stimulated HFD mouse aorta (PGF 2 𝛼, CTL, n = 7; clemizole, n = 5; TRPC5 − / − , n = 5; MAFP, n = 6; NS-398, n = 6; –endo, n = 6; PGE 2 , CTL, n = 7; clemizole, n = 5; TRPC5 − / − , n = 5; MAFP, n = 5; NS-398, n = 5; –endo, n = 5; (c) Effects of AM237 (100 nmol/L), MAFP (10 𝜇mol/L), and NS-398 (3 𝜇mol/L) treatment and the removal of endothelium (–Endo) on EIA for PGF 2 𝛼and PGE 2 in the NFD mouse aorta stimulated with ACh (10 𝜇mol/L) (PGF 2 𝛼, CTL, n = 7; AM237, n = 4; MAFP, n = 6; NS-398, n = 4; –endo, n = 4; PGE 2 , CTL, n = 10; AM237, n = 5; MAFP, n = 5; NS-398, n = 5; –endo, n = 7). (d) Schematic of the mechanism of TRPC5 regulation of endothelium-dependent contraction in the DIO mouse aorta. Mean ± SEM; a, ∗ P < 0.05, NS, no significant difference vs NFD, Student’s unpaired two-tailed t test; b, ∗ P < 0.05 vs CTL, one-way ANOVA followed by Dunnett’s multiple comparisons test; c, ∗ P < 0.05 vs CTL, # P < 0.05 vs AM237 group, one-way ANOVA followed by Turkey’s multiple comparisons test.

Article Snippet: The membranes were incubated overnight t 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), nti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and nti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidaseonjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beytime) at room temperature for 2 h. ImageJ was used for band intensity nalysis.

Techniques: Enzyme-linked Immunosorbent Assay, Knock-Out, Two Tailed Test

Journal: Journal of Integrative Agriculture

Article Title: Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4

doi: 10.1016/s2095-3119(17)61670-8

Figure Lengend Snippet: Fig. 1 Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 4 (nsp4) interacts with cytochrome c1 (cyto.c1). A, yeast two hybrid assay. The plasmids pGBKT7-nsp4 and pGADT7-cyto.c1 were co-transformed into the yeast strain Y2HGold and selected on QDO/X/ABA plates. The top and second panels are the respective positive and negative controls. B, HEK 293T cells were transfected to express Flag-cyto.c1, or HA-nsp4 or together. At 24 h post-transfection, the cells were either subjected to direct Western blot analysis or lysed and immunoprecipitated with anti-HA antibodies. The proteins bound to sepharose beads were separated by SDS-PAGE, transferred to PVDF membranes, and probed with the antibodies to Flag and HA. C, the same as B except that nsp4 truncation mutants were used. D, MARC-145 cells were transduced with letiviruses that were expressing GFP or nsp4-GFP in the presence of 8 µg mL–1 polybrene, respectively. The cells were harvested 48 h post-transduction and cell lysates were immunoprecipitated using anti-GFP beads. The proteins bound to sepharose beads were subjected to Western blot analysis using an anti-cytochrome c1 polyclonal antibody or anti-GFP mAb. E, PRRSV nsp4 interacts with cyto.c1 in mammalian cells. Vero cells grown on coverslips in six-well plates were transfected when 60–70% to express the indicated proteins, either alone or pairwise. At 18–24 h post-transfection, the cells were fixed, permeablized, and stained with antibodies to HA and Flag, and examined by confocal microscopy. For double transfections, the merged images are shown at the right.

Article Snippet: Mouse anti-green fluorescent protein (GFP) mAb (66002-1-Ig) and rabbit anti-cytochrome c1 polyclonal antibodies (10242-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Virus, Y2H Assay, Transformation Assay, Transfection, Western Blot, Immunoprecipitation, SDS Page, Transduction, Expressing, Staining, Confocal Microscopy