Journal: iScience

Article Title: Patient-derived tumor organoids mimic treatment-induced DNA damage response in glioblastoma.

doi: 10.1016/j.isci.2024.110604

Figure Lengend Snippet: Figure 1. Overview of the procedures used to generate GBOs from resected tumor tissue, cell-type-specific immunostaining of the GBOs and parental tumors and cytokine secretion from GBOs (A) Schematic presentation of main steps for generating GBOs from resected tumor tissue, created with bioRender.com. (B) Growth rate as the quantification of relative area of viable GBOs for five weeks. Data represent mean values GS.E.M. (n = 3 GBOs samples per patient (P)). (C) Immunofluorescence analyses of the GBO TME. GBOs express markers of GSCs (SOX2, CD9, and CD44) and TME cells, such as markers of cells of vasculature (CD31 and CD105), as well as macrophages and microglia (CD68 and Iba1). Representative images from a tissue sample from one patient are shown. Scale bar: 100 mm. (D) Selected cytokines and growth factors are secreted by GBOs in comparison to cultured GB cells from the same patient.

Article Snippet: Antibodies Mouse monoclonal to SOX2 Abcam Cat#ab171380; RRID:AB_2732072 Rabbit polyclonal to GFAP Abcam Cat#ab211271; RRID: N/A Mouse monoclonal to Iba1 Abcam Cat#ab15690; RRID:AB_2224403 Rabbit polyclonal to CD68 Atlas antibodies Cat#HPA048982; RRID:AB_2680587 Mouse monoclonal to CD44 BioRad Cat#MCA2504; RRID:AB_808430 Rabbit monoclonal to CD9 Cell signaling technology Cat#13403; RRID:AB_2732848 Mouse monoclonal to CD31 (PECAM-1) Cell signaling technology Cat#3528; RRID:AB_2160882 Rabbit polyclonal to CD105 (ENG) Atlas antibodies Cat#HPA067440; RRID:AB_2685840 Rabbit Monoclonal to CD3E Abcam Cat#ab16669; RRID:AB_443425 Mouse monoclonal to PD-1 Cell signaling technology Cat#43248S; RRID:AB_2728836 Rabbit Monoclonal to PD-L1 Cell signaling technology Cat#13684T; RRID:AB_2687655 Mouse monoclonal to MDM2 [2A10] Abcam Cat#ab16895; RRID:AB_2143534 Rabbit monoclonal to p21 [EPR362] Abcam Cat#ab109520; RRID:AB_10860537 Rabbit monoclonal to MDM2 (D1V27) Cell signaling technology Cat#86934; RRID:AB_2784534 Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Invitrogen Cat#A11008; RRID:AB_143165

Techniques: Immunostaining, Comparison, Cell Culture

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: PLoS ONE

Article Title: Necl-4/SynCAM-4 Is Expressed in Myelinating Oligodendrocytes but Not Required for Axonal Myelination

doi: 10.1371/journal.pone.0064264

Figure Lengend Snippet: A. Western immuneblotting of brain tissues (from P10 wild-type and Necl-4 homozygous mice) with anti-Necl-1, anti-Necl-2 and anti-Necl-3 antibodies. β-actin was the internal control. B. Statistical analysis on the relative expression level of Necl1, Necl2 and Necl3 with Student's t-test (n = 3. Necl1, p = 0.70. Necl2, p = 0.72. Necl3, p = 0.69). Error bar, standard deviation.

Article Snippet: 30 mg protein from control and mutant tissues was loaded for SDS-PAGE electrophoresis and subsequently detected with anti-Necl-1 (developed in Peking Union Medical University), anti-Necl-2 (Proteintech, Cat# 14335-1-AP), anti-Necl-3 (Abcam Inc, Cat# ab133393) and anti-Necl-4 (UC Davis/NIH NeuroMab Facility, Cat#73-247), and mouse anti-β-actin (Sigma, Cat# A5316) antibodies according to the standard protocol.

Techniques: Western Blot, Expressing, Standard Deviation