Journal: Molecular Medicine

Article Title: NAT10 induces N4-acetylcytidine modification of AdipoR1-mediated mitochondrial biogenesis against endothelial-to-mesenchymal transition in hypertension

doi: 10.1186/s10020-025-01321-3

Figure Lengend Snippet: High NAT10 and ac4C levels in hypertension groups compared to the control groups. WB assay and the quantitative analysis of NAT10 level in hypertensive mice descending thoracic aortic tissues ( A , B ; n = 3 ) , SHRs descending thoracic aortic samples ( A , C ; n = 3 ) and Ang II treated HUVECs ( A , D ; n = 3 ) . ( E) The ac4C level in hypertensive mice descending thoracic aortic tissues, SHRs descending thoracic aortic samples and Ang II treated HUVECs ( n = 3). ( F) Representative IF staining of NAT10 and CD31 in hypertensive mice descending thoracic aortic tissues, SHRs descending thoracic aortic samples and the control tissues. Fluorescence in green represents CD31, while fluorescence in red represents NAT10 and fluorescence in blue represents DAPI. Scale bar, 100 μm. (representative images; n = 6). Data are presented as mean ± SD. ** p < 0.01. Statistical tests were performed using unpaired two-tailed Student’s t-test

Article Snippet: The descending thoracic aortic sections were blocked with blocking buffer (3% bovine serum albumin in PBS) for 60 min and stained with primary antibodies containing NAT10 (Abcam, ab194297, rabbit, 1:500), CD31 (MCE, YA806, mouse, 1:100) and SM22α (Proteintech, 10493-1-AP, rabbit, 1:100) overnight at 4 °C and the corresponding Alexa-conjugated secondary antibody containing Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 488) (Abcam, ab150105, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 594) (Abcam, ab150080, 1:1000), Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 594) (Abcam, ab150108, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) (Abcam, ab150077, 1:1000) at room temperature for 1 h. The nuclei were counterstained with DAPI (C0065, Solarbio) for 5 min, and the fluorescence images were captured under a fluorescence microscope (Olympus).

Techniques: Control, Staining, Fluorescence, Two Tailed Test

Journal: Molecular Medicine

Article Title: NAT10 induces N4-acetylcytidine modification of AdipoR1-mediated mitochondrial biogenesis against endothelial-to-mesenchymal transition in hypertension

doi: 10.1186/s10020-025-01321-3

Figure Lengend Snippet: NAT10 overexpression inhibited endothelial dysfunction and EndMT in hypertension. ( A , B ) WB assay and the quantitative analysis of NAT10 level in HUVECs after Ang II stimulation ( n = 3). ( C ) ECs proliferation analysis between OE-NAT10 and OE-NC group after Ang II stimulation ( n = 3). ( D ) ECs migration analysis between OE-NAT10 and OE-NC group after Ang II stimulation by Transwell migration assay ( n = 3). Scale bar, 100 μm. ( E ) ECs angiogenesis analysis between OE-NAT10 and OE-NC group after Ang II stimulation by tube formation assay ( n = 3). Scale bar, 100 μm. ( F , G ) WB assay and the quantitative analysis of CD31, VE-cadherin, SM22α and N-cadherin levels in HUVECs after Ang II stimulation ( n = 3). ( H ) H&E staining of descending thoracic aortic sections and the relative wall thickness of each group ( n = 6). Scale bar, 100 μm. ( I ) Masson staining of each group and quantitative analysis of the fibrotic area ( n = 6). Scale bar, 100 μm. ( J ) IF staining of CD31 and SM22α levels in OE-NAT10 and OE-NC group ( n = 6). Fluorescence in red represents CD31, while fluorescence in green represents SM22α and fluorescence in blue represents DAPI. Scale bar, 100 μm. ( K , L ) WB assay of CD31, VE-cadherin, SM22α and N-cadherin levels in OE-NAT10 and OE-NC group ( n = 6). Data represented as mean ± SD from three independent experiments. ** p < 0.01. Statistical tests were performed using unpaired two-tailed Student’s t-test (B-G, L) and Mann–Whitney U test (H, I)

Article Snippet: The descending thoracic aortic sections were blocked with blocking buffer (3% bovine serum albumin in PBS) for 60 min and stained with primary antibodies containing NAT10 (Abcam, ab194297, rabbit, 1:500), CD31 (MCE, YA806, mouse, 1:100) and SM22α (Proteintech, 10493-1-AP, rabbit, 1:100) overnight at 4 °C and the corresponding Alexa-conjugated secondary antibody containing Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 488) (Abcam, ab150105, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 594) (Abcam, ab150080, 1:1000), Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 594) (Abcam, ab150108, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) (Abcam, ab150077, 1:1000) at room temperature for 1 h. The nuclei were counterstained with DAPI (C0065, Solarbio) for 5 min, and the fluorescence images were captured under a fluorescence microscope (Olympus).

Techniques: Over Expression, Migration, Transwell Migration Assay, Tube Formation Assay, Staining, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Medicine

Article Title: NAT10 induces N4-acetylcytidine modification of AdipoR1-mediated mitochondrial biogenesis against endothelial-to-mesenchymal transition in hypertension

doi: 10.1186/s10020-025-01321-3

Figure Lengend Snippet: NAT10 depletion induced endothelial dysfunction and EndMT in hypertension. (A, B) WB assay and the quantitative analysis of NAT10 level in HUVECs after Ang II stimulation ( n = 3). (C) ECs proliferation analysis between sh-NAT10 and sh-NC group after Ang II stimulation ( n = 3). (D) ECs migration analysis between sh-NAT10 and sh-NC group after Ang II stimulation ( n = 3). Scale bar, 100 μm. (E) ECs angiogenesis analysis between sh-NAT10 and sh-NC group after Ang II stimulation ( n = 3). Scale bar, 100 μm. (F, G) WB assay and the quantitative analysis of CD31, VE-cadherin, SM22α and N-cadherin levels in HUVECs after Ang II stimulation ( n = 3). (H) H&E staining of descending thoracic aortic sections and the relative wall thickness of each group ( n = 6). Scale bar, 100 μm. (I) Masson staining of each group and quantitative analysis of the fibrotic area ( n = 6). Scale bar, 100 μm. (J) IF staining of CD31 and SM22α levels in sh-NAT10 and sh-NC group ( n = 6). Fluorescence in red represents CD31, while fluorescence in green represents SM22α and fluorescence in blue represents DAPI. Scale bar, 100 μm. (K, L) WB assay of CD31, VE-cadherin, SM22α and N-cadherin levels in sh-NAT10 and sh-NC group ( n = 6). Data represented as mean ± SD from three independent experiments. ** p < 0.01. Statistical tests were performed using unpaired two-tailed Student’s t-test (B-G, L) and Mann–Whitney U test (H, I)

Article Snippet: The descending thoracic aortic sections were blocked with blocking buffer (3% bovine serum albumin in PBS) for 60 min and stained with primary antibodies containing NAT10 (Abcam, ab194297, rabbit, 1:500), CD31 (MCE, YA806, mouse, 1:100) and SM22α (Proteintech, 10493-1-AP, rabbit, 1:100) overnight at 4 °C and the corresponding Alexa-conjugated secondary antibody containing Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 488) (Abcam, ab150105, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 594) (Abcam, ab150080, 1:1000), Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 594) (Abcam, ab150108, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) (Abcam, ab150077, 1:1000) at room temperature for 1 h. The nuclei were counterstained with DAPI (C0065, Solarbio) for 5 min, and the fluorescence images were captured under a fluorescence microscope (Olympus).

Techniques: Migration, Staining, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Medicine

Article Title: NAT10 induces N4-acetylcytidine modification of AdipoR1-mediated mitochondrial biogenesis against endothelial-to-mesenchymal transition in hypertension

doi: 10.1186/s10020-025-01321-3

Figure Lengend Snippet: Remodelin induced endothelial dysfunction and EndMT in hypertension. ( A , B) WB assay and the quantitative analysis of NAT10 level in HUVECs after Ang II stimulation ( n = 3). ( C - E) ECs proliferation, migration and angiogenesis analysis between remodelin and control group after Ang II stimulation ( n = 3). Scale bar, 100 μm. ( F , G ) WB assay and the quantitative analysis of CD31, VE-cadherin, SM22α and N-cadherin levels in HUVECs after Ang II stimulation ( n = 3). ( H ) H&E staining of descending thoracic aortic sections and the relative wall thickness of remodelin and control group ( n = 6). Scale bar, 50 μm. ( I) Masson staining of remodelin and control group and quantitative analysis of the fibrotic area ( n = 6). Scale bar, 50 μm. ( J) IF staining of CD31 and SM22α levels in remodelin and control group ( n = 6). Fluorescence in red represents CD31, while fluorescence in green represents SM22α and fluorescence in blue represents DAPI. Scale bar, 50 μm. ( K , L) WB assay of CD31, VE-cadherin, SM22α and N-cadherin levels in remodelin and control group ( n = 6). Data represented as mean ± SD from three independent experiments. ** p < 0.01. Statistical tests were performed using unpaired two-tailed Student’s t-test (B-G, L) and Mann–Whitney U test (H, I)

Article Snippet: The descending thoracic aortic sections were blocked with blocking buffer (3% bovine serum albumin in PBS) for 60 min and stained with primary antibodies containing NAT10 (Abcam, ab194297, rabbit, 1:500), CD31 (MCE, YA806, mouse, 1:100) and SM22α (Proteintech, 10493-1-AP, rabbit, 1:100) overnight at 4 °C and the corresponding Alexa-conjugated secondary antibody containing Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 488) (Abcam, ab150105, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 594) (Abcam, ab150080, 1:1000), Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 594) (Abcam, ab150108, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) (Abcam, ab150077, 1:1000) at room temperature for 1 h. The nuclei were counterstained with DAPI (C0065, Solarbio) for 5 min, and the fluorescence images were captured under a fluorescence microscope (Olympus).

Techniques: Migration, Control, Staining, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Medicine

Article Title: NAT10 induces N4-acetylcytidine modification of AdipoR1-mediated mitochondrial biogenesis against endothelial-to-mesenchymal transition in hypertension

doi: 10.1186/s10020-025-01321-3

Figure Lengend Snippet: AdipoR1 is a downstream target of NAT10 in Ang II treated ECs. ( A , B) WB assay and the quantitative analysis of AdipoR1 level in HUVECs after Ang II stimulation ( n = 3). ( C) ECs proliferation analysis after Ang II stimulation ( n = 3). ( D) ECs migration analysis after Ang II stimulation ( n = 3). Scale bar, 100 μm. ( E ) ECs angiogenesis analysis after Ang II stimulation ( n = 3). Scale bar, 100 μm. ( F , G) WB assay and the quantitative analysis of CD31, VE-cadherin, SM22α and N-cadherin levels in HUVECs after Ang II stimulation ( n = 3). ( H) H&E staining of descending thoracic aortic sections and the relative wall thickness of each group ( n = 6). Scale bar, 100 μm. ( I) Masson staining of each group and quantitative analysis of the fibrotic area ( n = 6). Scale bar, 100 μm. ( J) IF staining of CD31 and SM22α levels in each group ( n = 6). Fluorescence in red represents CD31, while fluorescence in green represents SM22α and fluorescence in blue represents DAPI. Scale bar, 100 μm. ( K , L) WB assay of CD31, VE-cadherin, SM22α and N-cadherin levels in each group ( n = 6). Data represented as mean ± SD from three independent experiments. ** p < 0.01, # p < 0.05, ## p < 0.01. Statistical analysis was performed using t-test (unpaired, two-sided) between two groups or one-way ANOVA followed by Tukey’s multiple comparisons test for multiple-group comparisons

Article Snippet: The descending thoracic aortic sections were blocked with blocking buffer (3% bovine serum albumin in PBS) for 60 min and stained with primary antibodies containing NAT10 (Abcam, ab194297, rabbit, 1:500), CD31 (MCE, YA806, mouse, 1:100) and SM22α (Proteintech, 10493-1-AP, rabbit, 1:100) overnight at 4 °C and the corresponding Alexa-conjugated secondary antibody containing Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 488) (Abcam, ab150105, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 594) (Abcam, ab150080, 1:1000), Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 594) (Abcam, ab150108, 1:500), Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) (Abcam, ab150077, 1:1000) at room temperature for 1 h. The nuclei were counterstained with DAPI (C0065, Solarbio) for 5 min, and the fluorescence images were captured under a fluorescence microscope (Olympus).

Techniques: Migration, Staining, Fluorescence

Journal: Oxidative medicine and cellular longevity

Article Title: Human Tissue Kallikrein 1 Improves Erectile Dysfunction of Streptozotocin-Induced Diabetic Rats by Inhibition of Excessive Oxidative Stress and Activation of the PI3K/AKT/eNOS Pathway.

doi: 10.1155/2020/6834236

Figure Lengend Snippet: Figure 2: Verification of CSMC and EC from WTR and TGR and the expression of the hKLK1 in EC by immunofluorescence. Panel (a) shows the verification of CSMC through the expression of desmin (magnification: ×200). Panel (b) shows the verification of EC through the expression of CD31, and hKLK1 was only expressed in EC from TGR but not WTR (magnification: ×100). Panel (c) shows the cell purities of EC and CSMC from WTR and TGR through the bar graph. hKLK1: human tissue kallikrein 1; rKLK1: rat tissue kallikrein 1; WTR: wild-type rats; TGR: transgenic rats; EC: endothelial cell; CSMC: cavernous smooth muscle cell; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: Primary antibodies against hKLK1 (1 : 200; Sigma-Aldrich), CD31 (1 : 100, Boster), and desmin (1 : 200; Abcam) were used in this study.

Techniques: Expressing, Transgenic Assay