Journal: Experimental & Molecular Medicine

Article Title: Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway

doi: 10.1038/s12276-024-01297-w

Figure Lengend Snippet: a VSMCs were transfected with control siRNA or TERT siRNA (10 μM) for 48 h, pretreated with 200 μM OA for 24 h, and incubated with 2 mM Met for 1 h, after which the protein levels were analyzed by Western blotting. b Cellular senescence was assessed by SA-β-gal staining and bright-field microscopy (200x, scale bar = 100 μm). c After transfection, the protein levels were analyzed by Western blotting with TNF-α and ADRP antibodies and d the cells were stained with BODIPY493/503 (a green lipid stain) (scale bar = 30 μm). e After transfection, colocalization of TERT (green) and 53BP1 foci (red) was observed by confocal microscopy. Nuclei were stained with DAPI (blue). Magnified views of merged images showing details of the colocalization are shown in the lower series of panels (scale bar = 30 μm). f Western blotting analysis of contractile (elastin and α−SMA) and synthetic proteins (collagen I) in VSMCs. Representative results are shown, and the values are the means ± SEs of three independent experiments.

Article Snippet: Con siRNA and TERT siRNA were purchased from Santa Cruz (Delaware, CA, USA).

Techniques: Transfection, Control, Incubation, Western Blot, Staining, Microscopy, Confocal Microscopy

Journal: Experimental & Molecular Medicine

Article Title: Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway

doi: 10.1038/s12276-024-01297-w

Figure Lengend Snippet: a VSMCs were transfected with TERT siRNA, and then gelatinolytic activity (MMP-2) was determined by gelatin zymography. MMP-2 expression was determined by Western blotting. b After transfection, MMP-2 expression was analyzed by immunofluorescence under a confocal microscope (scale bar = 30 μm). c To investigate inflammatory cytokines, VSMCs were analyzed by immunoblotting with proinflammatory cytokine (IL-6) and anti-inflammatory cytokine (IL-10) antibodies. d IL-10 (green) and IL-6 (red) were analyzed by immunofluorescence under a confocal microscope. Nuclei were stained with DAPI (blue) (scale bar = 30 μm). Representative results are shown, and the values are the means ± SEs of three independent experiments.

Article Snippet: Con siRNA and TERT siRNA were purchased from Santa Cruz (Delaware, CA, USA).

Techniques: Transfection, Activity Assay, Zymography, Expressing, Western Blot, Immunofluorescence, Microscopy, Staining

Journal: Experimental & molecular medicine

Article Title: Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway.

doi: 10.1038/s12276-024-01297-w

Figure Lengend Snippet: Fig. 2 The regulation of TERT expression via the phosphorylation of AMPK and the PGC1-α pathway by metformin (Met). a VSMCs were pretreated with OA (200 μM/24 hr) and then incubated with Met (2 mM/1 h). Protein expression levels were analyzed by Western blotting. b VSMCs were subjected to immunoprecipitation (IP) using a PGC-1α antibody and then immunoblotted with antibodies recognizing p-Ser (571) and TERT. Total PGC-1α levels in whole-cell lysates were estimated by Western blotting. c Western blot analysis of TERT and p-AMPK (Ser485) expression in OA-pretreated VSMCs cotreated with Met, Compound C (CC, 10 μM/1 h) or SR-18292 (SR, 10 μM/24 h). d ChIP‒qPCR analysis of TERT with the PGC-1α antibody in OA and Met-treated VSMCs. Representative results are shown, and the values are the means ± SEs of three independent experiments.

Article Snippet: Con siRNA and TERT siRNA were purchased from Santa Cruz (Delaware, CA, USA).

Techniques: Expressing, Phospho-proteomics, Incubation, Western Blot, Immunoprecipitation

Journal: Experimental & molecular medicine

Article Title: Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway.

doi: 10.1038/s12276-024-01297-w

Figure Lengend Snippet: Fig. 3 Met-induced upregulation of TERT expression and telomerase activity. a After treatment with OA, Met, and CC or SR-18292, telomerase activity was assessed using a TRAP (telomeric repeat amplification) assay. b VSMC phenotypes were determined by indirect immunofluorescence staining with TERT (green) (1:200) and DAPI (blue) antibodies (scale bar = 30 μm). c Representative images of telomere immunofluorescence-telomere FISH (IF-FISH, green) in VSMCs. Nuclei were stained with DAPI (blue) (scale bar = 30 μm). d VSMCs were stimulated with OA and treated with Met and CC or SR-18292. Relative telomere lengths were determined by qPCR. Representative results are shown, and the values are the means ± SEs of three independent experiments.

Article Snippet: Con siRNA and TERT siRNA were purchased from Santa Cruz (Delaware, CA, USA).

Techniques: Expressing, Activity Assay, Staining

Journal: Experimental & molecular medicine

Article Title: Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway.

doi: 10.1038/s12276-024-01297-w

Figure Lengend Snippet: Fig. 4 Effect of Met following TERT knockdown during the OA-induced pathogenesis of atherosclerosis and on senescent phenotypes. a VSMCs were transfected with control siRNA or TERT siRNA (10 μM) for 48 h, pretreated with 200 μM OA for 24 h, and incubated with 2 mM Met for 1 h, after which the protein levels were analyzed by Western blotting. b Cellular senescence was assessed by SA-β-gal staining and bright-field microscopy (200x, scale bar = 100 μm). c After transfection, the protein levels were analyzed by Western blotting with TNF-α and ADRP antibodies and d the cells were stained with BODIPY493/503 (a green lipid stain) (scale bar = 30 μm). e After transfection, colocalization of TERT (green) and 53BP1 foci (red) was observed by confocal microscopy. Nuclei were stained with DAPI (blue). Magnified views of merged images showing details of the colocalization are shown in the lower series of panels (scale bar = 30 μm). f Western blotting analysis of contractile (elastin and α−SMA) and synthetic proteins (collagen I) in VSMCs. Representative results are shown, and the values are the means ± SEs of three independent experiments.

Article Snippet: Con siRNA and TERT siRNA were purchased from Santa Cruz (Delaware, CA, USA).

Techniques: Knockdown, Transfection, Control, Incubation, Western Blot, Staining, Microscopy, Confocal Microscopy

Journal: Experimental & molecular medicine

Article Title: Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway.

doi: 10.1038/s12276-024-01297-w

Figure Lengend Snippet: Fig. 5 Effect of metformin following TERT knockdown on the OA-induced inflammatory response and senescence-associated secretory phenotype. a VSMCs were transfected with TERT siRNA, and then gelatinolytic activity (MMP-2) was determined by gelatin zymography. MMP- 2 expression was determined by Western blotting. b After transfection, MMP-2 expression was analyzed by immunofluorescence under a confocal microscope (scale bar = 30 μm). c To investigate inflammatory cytokines, VSMCs were analyzed by immunoblotting with proinflammatory cytokine (IL-6) and anti-inflammatory cytokine (IL-10) antibodies. d IL-10 (green) and IL-6 (red) were analyzed by immunofluorescence under a confocal microscope. Nuclei were stained with DAPI (blue) (scale bar = 30 μm). Representative results are shown, and the values are the means ± SEs of three independent experiments.

Article Snippet: Con siRNA and TERT siRNA were purchased from Santa Cruz (Delaware, CA, USA).

Techniques: Knockdown, Transfection, Activity Assay, Zymography, Expressing, Western Blot, Microscopy, Staining

Journal: Experimental & molecular medicine

Article Title: Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway.

doi: 10.1038/s12276-024-01297-w

Figure Lengend Snippet: Fig. 7 The effects of metformin on TERT expression in advanced atherosclerotic plaques and the senescent phenotypes of high-fat diet- fed ApoE KO mice. a, b Expression levels of p-AMPK (Ser485), PGC-1α, TERT, p53, and β-actin as determined by Western blotting of aortic tissues from HFD-fed mice (n = 4). c Immunofluorescence staining and colocalization of TERT (green) and α−SMA (red, a VSMC marker) in fixed tissue sections of atherosclerotic lesions from ApoE KO mice (n = 3, Scale bar = 30 μm). d After the mice were fed an HFD for 16 weeks, telomerase activity was assessed using a TRAP (telomeric repeat amplification protocol) assay in whole aortas from ApoE KO mice (n = 3, same aorta tissue used for DNA analysis). e Relative telomere lengths were determined by qPCR (n = 3, same aorta tissue for DNA analysis) using DNA samples from whole aortas of ApoE KO mice fed an HFD. f Expression levels of TNF-α, ADRP, and α-actin as determined by Western blotting of aortas after HFD consumption (n = 4). g Immunofluorescence staining and colocalization of BODIPY493/503-based lipid staining (green) and α-SMA (red, a VSMC marker) in fixed tissue sections of atherosclerotic lesions from ApoE KO mice (scale bar = 30 μm). Representative results are shown, and the values are the means ± SEs.

Article Snippet: Con siRNA and TERT siRNA were purchased from Santa Cruz (Delaware, CA, USA).

Techniques: Expressing, Western Blot, Staining, Marker, Activity Assay

Journal: Tumor Biology

Article Title: Effects of in vitro short- and long-term treatment with telomerase inhibitor in U-251 glioma cells

doi: 10.3233/tub-211515

Figure Lengend Snippet: Fig. 3. Effects of long-term exposure to MST-312 on cell morphology, telomerase expression and resistance to cytotoxic effects of the compound. (A) Dramatic changes on cell morphology, accessed by polarity index quantification, during the responding phase of long-term treatment. After 200 days of treatment, no difference on this morphological parameter between control and treated group was found (∗∗∗two-tailed p value < 0.001; Mann-Whitney test). (B) The treatment did not change levels of TERT mRNA. (C) Phase contrast micrographs show the resistance of long term MST-312 treated cells to cytotoxic effects of the drug at high concentration, which remains as characteristic of the culture at the end of experiment, showed by trypan blue exclusion assay (D).

Article Snippet: U-251 cells were transfected with TERT shRNA plasmid (sc-36641-SH, Santa Cruz Biotechnology®), using the Lipofectamine 3000 protocol, submitted to selection with puromycin for five days and then grown until reach 70% confluence.

Techniques: Expressing, Control, MANN-WHITNEY, Concentration Assay, Trypan Blue Exclusion Assay

Journal: Tumor Biology

Article Title: Effects of in vitro short- and long-term treatment with telomerase inhibitor in U-251 glioma cells

doi: 10.3233/tub-211515

Figure Lengend Snippet: Fig. 6. Toxic effect of MST-312 on TERT knock-down U-251 cells. (A) Characterization of knock-down cells by qPCR showing low TERT expression in TERT -shRNA plasmid transfected cells compared to control-shRNA plasmid transfected cells. (B) 72 h dose-response curve (MTT assay, the control is not shown due to the use of log of concentrations - basal viability values in both control and KD groups were comparable) showing that TERT knock-down cells are more resistant to MST-312 effects, which was also clearly demonstrated by phase-contrast microscopy analysis (C). (D) In addition, clonal cultures with high TERT expression exhibited greater susceptibility to high concentration of the drug (50 M) (72 h treatment; ∗two-tailed p value < 0.05; Mann-Whitney test).

Article Snippet: U-251 cells were transfected with TERT shRNA plasmid (sc-36641-SH, Santa Cruz Biotechnology®), using the Lipofectamine 3000 protocol, submitted to selection with puromycin for five days and then grown until reach 70% confluence.

Techniques: Knockdown, Expressing, shRNA, Plasmid Preparation, Transfection, Control, MTT Assay, Microscopy, Concentration Assay, MANN-WHITNEY