Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: STX6 is a target of miR‐375‐3p in endothelial cell senescence. (a) Predicted and mutated miR‐375‐3p binding sites within the 3′UTR of STX6. (b) Luciferase assay of WT and mutant STX6 3′UTR reporters in HEK293A cells transfected with miR‐375‐3p mimic or scramble. (c) qRT‐PCR analysis of STX6 mRNA in HUVECs transfected with miR‐375‐3p mimic or scramble, normalized to GAPDH. (d, e) Western blot analysis of STX6 protein expression in HUVECs transfected with miR‐375‐3p mimic (d) or STX6 siRNAs (e), normalized to GAPDH. (f) Representative SA‐β‐gal staining and quantification in HUVECs transfected with STX6 siRNAs or scramble. Scale bar = 200 μm. (g) Representative colony formation images showing cell proliferation in HUVECs transfected with STX6 siRNAs or scramble. Scale bar = 5 mm. (h) qRT‐PCR analysis of p15, IL6, and IL8 mRNA in HUVECs transfected with STX6 siRNAs or scramble, normalized to GAPDH. Data are presented as mean ± SD; statistical significance determined by unpaired two‐tailed Student's t ‐test. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Binding Assay, Luciferase, Mutagenesis, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Staining, Two Tailed Test

Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: STX6 overexpression rescues the pro‐senescent effect of miR‐375‐3p. (a) Representative SA‐β‐gal staining and quantification in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p mimic. Scale bar = 200 μm. (b) qRT‐PCR analysis of p15, IL6, and IL8 mRNA expression in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p mimic, normalized to GAPDH. (c) Colony formation assay showing cell proliferation in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p mimic. Scale bar = 5 mm. Data are presented as mean ± SD; statistical significance determined by two‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Over Expression, Staining, Infection, Transfection, Quantitative RT-PCR, Expressing, Colony Assay

Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: miR‐375‐3p/STX6 signaling promotes endothelial cell senescence through the increased internalization of TGFBR1. (a) Immunofluorescence images showing colocalization (yellow) of TGFBR1 (green) and EEA1 (red) in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p. Nuclei stained with Hoechst (blue). Scale bar = 5 μm. (b–d) Western blot analysis of SMAD2 phosphorylation and p15 expression in HUVECs transfected with miR‐375‐3p mimic or STX6 siRNAs (b), infected with Ad‐STX6 and transfected with miR‐375‐3p mimic (c), or co‐transfected with SMAD2 siRNA and miR‐375‐3p mimic or STX6 siRNAs (d). (e) Representative SA‐β‐gal staining and quantification in HUVECs co‐transfected with SMAD2 siRNA and miR‐375‐3p mimic or STX6 siRNAs. Scale bar = 200 μm. (f, g) Western blot (f) and SA‐β‐gal (g) staining in HUVECs infected with Ad‐STX6 and stimulated with TGF‐β1. Scale bar = 200 μm. Data are presented as mean ± SD; statistical significance determined by unpaired two‐tailed Student's t ‐test or two‐way ANOVA. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Immunofluorescence, Infection, Transfection, Staining, Western Blot, Phospho-proteomics, Expressing, Two Tailed Test

Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: Overexpression of STX6 reduce atherosclerotic plaque in mice. (a) qRT‐PCR analysis of Stx6 expression in aortas of WT ( n = 21) and ApoE −/− ( n = 23) mice fed HFD for 8 weeks, normalized to Gapdh. (b) Schematic of experimental design showing Ad‐Ctrl or Ad‐STX6 injection (1 × 10 9 PFU per mouse, twice weekly for 8 weeks) in ApoE −/− mice on HFD. (c) Representative Oil Red O staining images and quantification of lesion size and positive area in the aortic sinus. Scale bar = 200 μm. (d) Representative Oil Red O staining images and quantification of plaque area in the entire aortic tree. (e) Immunofluorescence staining of macrophage marker MOMA2 (green) in aortic sinus. Scale bar = 200 μm. (f) Representative SA‐β‐gal and Cd31 co‐staining in aortic sinus lesions. Scale bar = 5 μm. (g) Immunofluorescence of phosphorylated Smad2 (green) and Cd31 (red) in the aortic intima (L). Nuclei stained with Hoechst (blue). Scale bar = 5 μm. Data are presented as mean ± SD; statistical significance determined by unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Injection, Staining, Immunofluorescence, Marker, Two Tailed Test