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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 1. Comparison of NPRA and NPRC protein expression. (A) Protein expression of NPRA and NPRC were visualized by western blotting in membrane enriched fractions prepared from a variety of mouse tissues: gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), heart, liver, kidney, and lung. (B) BAT membrane enriched fractions from wildtype and NPRC−/− mice fed either standard chow or HFD were used to visualize NPRA and NPRC protein levels. Šídák’s multiple comparisons test for two-way ANOVA indicated on graph. * P < 0.05, *** P < 0.001, **** P < 0.0001. (C) BAT protein from chow- and HFD-fed mice was separated into membrane and cytosolic enriched fractions. NPRA and NPRC were assessed by western blotting. (D) HIB-1B cells were transfected with the indicated plasmids and treated with adipocyte differentiation cocktail for 48 h. Cells were then treated with the 2.5 µM MG-132 for 16 h. Flag-NPRA and NPRC were assessed from whole-cell lysates by western blotting. Šídák’s multiple comparisons test for one-way ANOVA indicated on graph. ** P < 0.01, *** P < 0.001.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Comparison, Expressing, Western Blot, Membrane, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 2. NPRC immunoprecipitates with NPRA and NPRB. HEK 293FT cells were transfected with plasmids expressing Flag-tagged natriuretic peptide receptors. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag Affinity agarose. (A) Endogenous NPRC coimmunoprecipitated with hNPRA-Flag, hNPRB-Flag, or VEGFR1-Flag. (B) Endogeneous NPRC coimmunoprecipitated with GC-C-Flag. (C) NPRC band intensity to input of NPRC was calculated through Image J software was presented in the right panel. Šídák’s multiple comparisons test for one-way ANOVA comparing all samples to the negative control VEGFR1 for western blots in A and B are indicated on graph. *P < 0.05, ** P < 0.01, *** P < 0.001. (D) hNPRA-Flag expressing HEK 293FT cells were treated with ANP (ANP1-28, 200 nM) or a truncated form of ANP that preferentially binds NPRC (ANP4-23, 200 nM) for 30 min. Neither ANP1-28 or ANP4-23 affected the ability of endogenous NPRC to coimmunoprecipitate with hNPRA-Flag. (E) Endogenous NPRC coimmunoprecipitated with Flag-rNPRA and a truncated version of Flag- rNPRA lacking the c-terminal amino acids 528-1057 [Flag-rNPRA(1-527)].

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Transfection, Expressing, Immunoprecipitation, Software, Negative Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 3. Endogenous natriuretic peptide receptor expression in cell models used. Expression of NPRA, NPRB, and NPRC were detected by western blot in HeLa, HEK293FT, and HAP-1 cells. β-actin was used as the loading control.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Expressing, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 6. Coexpression of NPRC reduces ANP-evoked cGMP production in plasma membranes. Plasma membranes were made from HEK-293T cells transfected with either NPRA-flag plus pcDNA3-Clover control or NPRA-flag plus NPRC-flag. Plasma membranes were made without or with (SI Appendix, Fig. S3) phosphatase inhibitors. Co-expression of hNPRC along with hNPRA reduced the cGMP generated in the presence of 100 nM ANP (P = 0.0163, test for difference in slopes between NPRA and NPRA+NPRC; Šídák’s multiple comparisons test comparing NPRA vs. NPRA+NPRC are indicated on the graphs, ** P < 0.01.). Untransfected cells did not appear to generate cGMP. Western blot of membranes used in the ANP-stimulated cGMP assay.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Clinical Proteomics, Transfection, Control, Expressing, Generated, Western Blot