Journal: Theranostics

Article Title: Cardiomyocyte-derived OTUD7B promotes cardiac hypertrophy by deubiquitinating SERCA2a

doi: 10.7150/thno.129105

Figure Lengend Snippet: Cardiomyocyte-specific OTUD7B overexpression promotes cardiac hypertrophy by deubiquitinating SERCA2a at K628. (A) Schematic diagram of the TAC-induced mouse model. WT mice were injected with AAV9 cardiomyocyte-specific overexpressing SERCA2a WT or SERCA2a K628R , and OTUD7B OE or empty vector (EV) by the tail vein. After 2 weeks, mice were subjected to TAC. After 4 weeks, the cardiac function of mice was assessed using echocardiography. Mice were then euthanized, and samples were harvested. (B) Representative left ventricular M-mode echocardiographic images. (C-D) Values of ejection fraction (C) and fractional shortening (D). EF, ejection fraction; FS, fractional shortening; n = 6. (E) Representative whole heart images. (F) The ratio of heart weight to body weight (HW/BW). n = 6. (G) The ratio of heart weight to tibial length (HW/TL). n = 6. (H) Representative HE-stained images of cardiac tissue sections. (I-J) Representative images (I) and quantification (J) of wheat germ agglutinin (WGA)-stained cardiac tissue sections (n = 6). (K-L) Representative images (K) and quantification (L) from Masson's trichrome-stained cardiac tissue sections (n = 6). (M) Serum atrial natriuretic peptide (ANP) levels were detected using ELISA kits (n = 6). (N-P) mRNA levels of Myh7 (N), Nppa (O), and Nppb (P) in heart tissues (n = 6). Signal intensities were adjusted using Actb as the normalization control.

Article Snippet: Animals were maintained for 4 weeks after TAC or sham surgery. (3) OTUD7B (OTUD7B OE ) and SERCA2a (SERCA2a WT or SERCA2a K628R ) cardiomyocyte-specific overexpression was achieved using recombinant adeno-associated virus serotype 9 (AAV9) vectors driven by the cTNT promoter (Genechem, Shanghai, China).

Techniques: Over Expression, Injection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature Communications

Article Title: Ras-related protein 2 limits vascular smooth muscle cell phenotypic switching and abdominal aortic aneurysm development

doi: 10.1038/s41467-025-64496-7

Figure Lengend Snippet: a Volcano plot of deregulated transcripts in HASMCs stimulated with Ang II compared with PBS. The p-value was calculated by a Wilcoxon rank sum test. No correction was applied for multiple testing. b MEG3 expression level in aorta of AAA patients and healthy control individuals (non-AAA). n = 6 distinct samples for each group, two-sided Unpaired t -text. c Meg3 expression level in aorta of Sham and AAA mice. n = 5 mice for each group, two-sided Mann–Whitney test. d Fluorescence in situ hybridization (FISH) assay to determine the expression of MEG3 in aortic sections from AAA patients and non-AAA. Scale bar: 50 μm. MEG3: Red, DAPI: Blue. 3 independent experiments showed consistent results. e Fluorescence in situ hybridization (FISH) assay to determine the expression of MEG3 in HASMCs simulated with Ang II. Scale bar: 50 μm. MEG3 : Red, DAPI: Blue. 3 independent experiments showed consistent results. f Representative western blotting and quantification of R-Ras2 in HASMCs transfected with si- MEG3 . n = 6 distinct samples for each group, two-sided unpaired t -test. g Representative western blotting and quantification of R-Ras2 in HASMCs overexpressing MEG3 and stimulated with Ang II. n = 6 distinct samples for each group. Two-way ANOVA test followed by Turkey’s multiple comparisons test. h HASMCs were overexpressed with MEG3, followed by transfected with si- RRAS2 and stimulated with Ang II. Protein levels of Calponin 1, α-SMA, SM22, and R-Ras2 were determined and their relative expression levels were analyzed. n = 6 distinct samples for each group. Two-way ANOVA test followed by Turkey’s multiple comparisons test. i Representative image of in situ zymography for detection of MMPs activity (Green). DAPI: Blue. Scale bar = 50 μm. n = 6 distinct samples for each group. Two-way ANOVA test followed by Turkey’s multiple comparisons test. All data were represented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors containing MEG3 driven by murine SM22a promoter, or control vector (AAV9-pCMV- MEG3 or AAV9-pCMV- vector ) were constructed by GeneChem Co., Ltd (Shanghai, China).

Techniques: Expressing, Control, MANN-WHITNEY, Fluorescence, In Situ Hybridization, Western Blot, Transfection, In Situ, Zymography, Activity Assay

Journal: Nature Communications

Article Title: Ras-related protein 2 limits vascular smooth muscle cell phenotypic switching and abdominal aortic aneurysm development

doi: 10.1038/s41467-025-64496-7

Figure Lengend Snippet: a Identification of ELAVL 1 as RRAS2 and MEG3 binding protein. b RRAS2 mRNA stability in HASMCs transfected with si- ELAVL 1 . n = 3 distinct samples. c Interaction of MEG3 with ELAVL 1 determined by RNA pull-down assay. d Interactions of ELAVL1 with full-length or MEG3 truncations. e Interaction between MEG3 and ELAVL 1 determined by RIP assay. n = 5 distinct samples, two-sided Unpaired t -test with Welch’s correction. f The interaction between RRAS2 and ELAVL 1. n = 5 distinct samples, two-sided Unpaired t -test. g Protein level of R-Ras2 in HASMCs transfected with si- ELAVL 1 . n = 6 distinct samples, two-sided Unpaired t -test with Welch’s correction. h Expression of R-Ras2 in HASMCs overexpressing with ELAVL1 and stimulated with Ang II. n = 6 distinct samples for each group. Two-way ANOVA test followed by Turkey’s multiple comparisons test. i HASMCs were overexpressed with MEG3 , followed by transfected with si-ELAVL 1 and stimulated with Ang II, the level of RRAS2 were determined. n = 6 independent experiments. Two-way ANOVA test followed by Turkey’s multiple comparisons test. j Schematic of 3′UTR, CDS and 5′UTR of RRAS2 reporter plasmids. k , l Luciferase activity of RRAS2 reporter plasmids. n = 6 independent experiments for each group, two-sided Unpaired t -test. m Schematic prediction of ELAVL 1 binding AU-rich elements (AREs) within the 3′UTR of RRAS2 . n Luciferase activity of RRAS2 3′UTR variants (Full, a – e ) in HEK293 cells with empty or ELAVL1 overexpressing vector. n = 6 independent experiments for each group, two-sided Unpaired t -test. o RIP analysis of relative enrichment of RRAS2 transcripts in ELAVL 1-immunoprecipitates. n = 5 independent experiments for each group, two-sided Unpaired t -test with Welch’s correction. All data were represented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors containing MEG3 driven by murine SM22a promoter, or control vector (AAV9-pCMV- MEG3 or AAV9-pCMV- vector ) were constructed by GeneChem Co., Ltd (Shanghai, China).

Techniques: Binding Assay, Transfection, Pull Down Assay, Expressing, Luciferase, Activity Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Ras-related protein 2 limits vascular smooth muscle cell phenotypic switching and abdominal aortic aneurysm development

doi: 10.1038/s41467-025-64496-7

Figure Lengend Snippet: a Representative photograph of aortas in each group. b Incidence of AAA. AAV vector + Ang II ( n = 12) and AAV MEG3 + Ang II ( n = 11), two-sided Fisher’s exact test. N.D. means Not Detected. c Quantification of the maximal abdominal aorta lumen diameters in each group at different time points. Two-way RM ANOVA followed by Bonferroni’s multiple comparisons test. d Ratio of aortic weight to body weight. Two-way ANOVA test followed by Turkey’s multiple comparisons test. c , d : AAV vector + Saline ( n = 5), AAV vector + Ang II ( n = 8), AAV MEG3 + Saline ( n = 5) and AAV MEG3 + Ang II ( n = 9). e Western blot and quantification of α-SMA, Calponin 1, and SM22 in aortic tissue of mice subjected with different treatment. AAV vector + Saline ( n = 5), AAV vector + AngII ( n = 6), AAV MEG3 + Saline ( n = 5) and AAV MEG3 + Ang II ( n = 6). Two-way ANOVA test followed by Turkey’s multiple comparisons test. f Representative H&E staining, Masson, and EVG staining of supra-renal abdominal aorta. Scale bar = 50 μm. Collagen content, AAV vector + Saline ( n = 5), AAV vector + AngII ( n = 6), AAV MEG3 + Saline ( n = 5) and AAV MEG3 + Ang II ( n = 6). Two-way ANOVA test followed by Turkey’s multiple comparisons test, EVG elastin degradation score, AAV vector + Saline ( n = 5), AAV vector + Ang II ( n = 8), AAV MEG3 + Saline ( n = 5), and AAV MEG3 + Ang II ( n = 9), two-sided Mann–Whitney test. g Representative immunofluorescent staining of MMPs activity determined by in situ zymography. Scale bar: 20 μm. AAV vector + Saline ( n = 5), AAV vector + AngII ( n = 6), AAV MEG3 + Saline ( n = 5) and AAV MEG3 + Ang II ( n = 6). Two-way ANOVA test followed by Turkey’s multiple comparisons. Elastin degradation score in ( f ) was represented as the box plots indicating median (middle line), 25th, 75th percentile (box), as well as minimum and maximum values (whiskers). The left data were represented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors containing MEG3 driven by murine SM22a promoter, or control vector (AAV9-pCMV- MEG3 or AAV9-pCMV- vector ) were constructed by GeneChem Co., Ltd (Shanghai, China).

Techniques: Plasmid Preparation, Saline, Western Blot, Staining, MANN-WHITNEY, Activity Assay, In Situ, Zymography