Journal: Aging and Disease

Article Title: The NRF2/ID2 Axis in Vascular Smooth Muscle Cells: Novel Insights into the Interplay between Vascular Calcification and Aging

doi: 10.14336/AD.2024.0075

Figure Lengend Snippet: Age Exacerbated VC and Reduced NRF2 Signal in Mice . ( A ) Representative micrographs depict von Kossa staining in serial sections of aged and young mice aortas under normal or high-phosphate (High-Pi) conditions (Scale bars represent 250μm and 50μm). ( B ) Quantification of calcium deposition in the aortas of aged and young mice with or without High-Pi stimulation (n=6, 10, 6, and 10, respectively). ( C ) Immunofluorescence co-staining illustrates P16 (red) and α-SMA (α-smooth muscle actin; green) in serial sections of aged and young mice aortas under both conditions (Scale bars represent 100μm, 50μm and 20μm). Arrows indicate areas of intranuclear P16 upregulation. ( D-E ) Representative micrographs of TUNEL assay (Scale bars represent 100μm and 50μm) and percentage of TUNEL-positive cells in serial sections of aged and young mice aortas, with or without High-Pi stimulation (n=6 per group). ( F ) Western blot analysis of cleaved-Caspase3 and Caspase3 in aged and young mice aortas with or without High-Pi stimulation. ( G ) Western blot analysis of Nrf2 in aged and young mice aortas. ( H-I ) Quantification of western blot results of cleaved-Caspase3 and Caspase3 in aged and young mice aortas with or without High-Pi stimulation (relative to β-Tubulin, n=4 per group). ( J ) Quantitative real-time PCR results of Nrf2 in aged and young mice aortas (relative to β-actin, n=6 per group). ( K ) Quantification of Western blot results of Nrf2 in aged and young mice aortas (relative to GAPDH, n=4 per group). ( L ) Immunohistochemistry staining displaying Nrf2 in serial sections of aged and young mice aortas (Scale bars represent 400μm and 100μm). ( M-N ) Calcium deposition quantified in High-Pi induced aged mice aortas with or without NRF2 activator (DMF or tBHQ) intervention (n=6 per group). ‘C’ stands for ‘under normal condition’, ‘PB’ stands for ‘under high-phosphate condition’.

Article Snippet: The primary antibodies were: α-smooth muscle actin (α-SMA) (BM0002, 1:200, BOSTER, China), ID2 (3431, 1:70, CST, USA), p16 (ab54210, 1:100, Abcam, USA), and Nrf2 (GTX103322, 1:500, GeneTex, USA).

Techniques: Staining, Immunofluorescence, TUNEL Assay, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: Aging and Disease

Article Title: The NRF2/ID2 Axis in Vascular Smooth Muscle Cells: Novel Insights into the Interplay between Vascular Calcification and Aging

doi: 10.14336/AD.2024.0075

Figure Lengend Snippet: NRF2 Deficiency in VSMC Contributes to Vitamin D-mediated VC and VSMC Senescence . ( A ) Representative photographs of Alizarin Red S staining in the aortas of Nrf2 SMCKO and Nrf2 WT mice following injection with dextrose water or vitamin D, scale bars represent 5mm. ( B ) Representative photographs of von Kossa staining in serial sections of Nrf2 SMCKO and Nrf2 WT mice aortas post-injection with dextrose water or vitamin D, scale bars represent 200μm and 50μm. ( C ) Quantitative analysis of calcium deposition in the aortas of Nrf2 SMCKO and Nrf2 WT mice (n=8 per group). ( D ) Representative photographs of BMP2 immunohistochemistry staining in serial sections of Nrf2 SMCKO and Nrf2 WT mice aortas after dextrose water or vitamin D injection, scale bars represent 400μm and 50μm. ( E ) Quantitative real-time PCR for Nrf2, Nqo1, Bmp2, and Runx2 in the aortas of Nrf2 SMCKO and Nrf2 WT mice treated with dextrose water or vitamin D (relative to β-actin, n=6 per group). ( F ) Representative micrographs of DHE staining in serial sections of Nrf2 SMCKO and Nrf2 WT mice aortas injected with dextrose water or vitamin D, scale bars represent 100μm and 50μm. ( G ) Immunofluorescence staining for P16 (red) and α-SMA (green) in serial sections of Nrf2 SMCKO and Nrf2 WT mouse aortas injected with dextrose water or vitamin D, scale bars represent 100μm, 50μm and 20μm. Arrows indicate areas of intranuclear P16 upregulation. ( H ) Quantitative analysis of the percentage of DHE-positive areas in the aortas of Nrf2 SMCKO and Nrf2 WT mice treated with dextrose water or vitamin D (n=6 per group). ( I-J ) Quantitative real-time PCR for p16, p21, and SASP components (Il-6, Il-8, Ccl2, Cxcl1, Csf2, Opg) in the aortas of Nrf2 SMCKO and Nrf2 WT mice following dextrose water or vitamin D injection (relative to β-actin, n=6 per group). Scale bars represent 100μm, 50μm and 20μm. ‘vD’ stands for ‘vitamin D treatment’.

Article Snippet: The primary antibodies were: α-smooth muscle actin (α-SMA) (BM0002, 1:200, BOSTER, China), ID2 (3431, 1:70, CST, USA), p16 (ab54210, 1:100, Abcam, USA), and Nrf2 (GTX103322, 1:500, GeneTex, USA).

Techniques: Staining, Injection, Immunohistochemistry, Real-time Polymerase Chain Reaction, Immunofluorescence