Journal: Oxidative Medicine and Cellular Longevity

Article Title: RRP Regulates Autophagy through the AMPK Pathway to Alleviate the Effect of Cell Senescence on Atherosclerosis

doi: 10.1155/2023/9645789

Figure Lengend Snippet: Activation of AMPK pathway by RRP promotes autophagy delaying vascular senescence. (a) Detection of AMPK α and m-TOR levels in total vascular proteins of mice by western blot. Data are shown as mean values ± SD per group and expressed as fold-over the control mean. (b) The amounts of AMPK α and m-TOR in the mRNA of mouse vasculature were detected by qRT-PCR in each group. Data are shown as mean values ± SD per group and expressed as fold-over the model mean. (c) Levels of autophagy-related proteins p62, beclin-1, and LC3B in total vascular proteins of mice. Data are shown as mean values ± SD of three animals per group and expressed as fold-over the control mean ( ∗ p < 0.05 and ∗∗ p < 0.001 vs. control; # p < 0.05 and ## p < 0.001 vs. model by one-way ANOVA test).

Article Snippet: The following primary antibodies were used: p16 (1 : 1000, cat. no. 18769, CST), beclin-1 (1 : 1000, cat. no. 3495, CST), NF- κ B p65 (1 : 1000, cat. no. 8242, CST), AMPK α (1 : 1000, cat. no. 2532, CST), p-AMPK α (1 : 1000, cat. no. 50081, CST), m-TOR (1 : 1000, cat. no. 2983, CST), p-mTOR (1 : 1000, cat. no. 5536, CST), p53 (1 : 1000, cat. no. 2524, CST), p21 (1 : 1000, cat. no. ab109199, Abcam), gamma-H2AX (1 : 5000, cat. no. ab81299, Abcam), eNOS (1 : 1000, cat. no. 32027, CST), p62 (1 : 1000, cat. no. ab91526, Abcam), lc3B (1 : 1000, cat. no. 43566, CST), and β -actin (1 : 1000, cat. no. 20536-1-AP, Proteintech).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: RRP Regulates Autophagy through the AMPK Pathway to Alleviate the Effect of Cell Senescence on Atherosclerosis

doi: 10.1155/2023/9645789

Figure Lengend Snippet: RRP delays aging by activating the AMPK pathway. (a) The levels of AMPK α , p-AMPK α , m-TOR, and p-m-TOR in the total protein of each HAECS group were detected by western blot. Data are shown as mean values ± SD per group and expressed as fold-over the control mean. (b) The amounts of AMPK α and m-TOR in mRNA of each HAECS group were detected by qRT-PCR. Data are shown as mean values ± SD per group and expressed as fold-over the H 2 O 2 mean ( ∗ p < 0.05 and ∗∗ p < 0.001 vs. control; # p < 0.05 and ## p < 0.001 vs. H 2 O 2 by one-way ANOVA test).

Article Snippet: The following primary antibodies were used: p16 (1 : 1000, cat. no. 18769, CST), beclin-1 (1 : 1000, cat. no. 3495, CST), NF- κ B p65 (1 : 1000, cat. no. 8242, CST), AMPK α (1 : 1000, cat. no. 2532, CST), p-AMPK α (1 : 1000, cat. no. 50081, CST), m-TOR (1 : 1000, cat. no. 2983, CST), p-mTOR (1 : 1000, cat. no. 5536, CST), p53 (1 : 1000, cat. no. 2524, CST), p21 (1 : 1000, cat. no. ab109199, Abcam), gamma-H2AX (1 : 5000, cat. no. ab81299, Abcam), eNOS (1 : 1000, cat. no. 32027, CST), p62 (1 : 1000, cat. no. ab91526, Abcam), lc3B (1 : 1000, cat. no. 43566, CST), and β -actin (1 : 1000, cat. no. 20536-1-AP, Proteintech).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: RRP Regulates Autophagy through the AMPK Pathway to Alleviate the Effect of Cell Senescence on Atherosclerosis

doi: 10.1155/2023/9645789

Figure Lengend Snippet: siAMPK α inhibited the protective effect of RRP on the autophagic function of HAECs. (a) siAMPK α intervention in HAECs for 8 h and AMPK α amounts in mRNA of HAECs after 2 d. (b) DCFH-DA detection of ROS levels in each group of HAECs after siAMPK α treatment. Scale bar = 100 μ m. (c) Levels of AMPK α , p-AMPK α , mTOR, p-mTOR, eNOS, p62, beclin-1, and LC3B in siAMPK α , H 2 O 2 , RRP, Met-treated, or untreated HAECs were detected by western blot. Data are shown as mean values ± SD per group and expressed as fold-over the control mean. (d) Detection of mtROS levels in each group by MitoSOX Red staining after siAMPK α treatment. Scale bar = 50 μ m. (e) Detection of mitochondrial membrane potential by JC-1 probe after siAMPK α treatment in HAECs. Scale bar = 100 μ m. Data are shown as mean values ± SD. (f) Transmission electron microscopy to detect the change of autophagic vesicles in each group of HAECs. (g) Levels of OCR profile and maximal respiration, basal respiration, and ATP rate in HAECs after siAMPK α treatment by transmission electron microscopy to observe the levels of autophagic vesicles in HAECs after siAMPK α treatment. Data are shown as mean values ± SD. (h) Laser confocal microscopy observation of adenovirus-transduced mRFP-GFP LC3B levels in HAECs after siAMPK α treatment. Scale bar = 10 μ m. (i) Detection of AMPK α and mTOR levels in siAMPK α -treated HAECs by qRT-PCR. Data are shown as mean values ± SD per group and expressed as fold-over the H 2 O 2 mean ( ∗ p < 0.05 and ∗∗ p < 0.001 vs. control; # p < 0.05 and ## p < 0.001 vs. siAMPK α +H 2 O 2 ; ♢ = ns vs. siAMPK α +H 2 O 2 by one-way ANOVA test).

Article Snippet: The following primary antibodies were used: p16 (1 : 1000, cat. no. 18769, CST), beclin-1 (1 : 1000, cat. no. 3495, CST), NF- κ B p65 (1 : 1000, cat. no. 8242, CST), AMPK α (1 : 1000, cat. no. 2532, CST), p-AMPK α (1 : 1000, cat. no. 50081, CST), m-TOR (1 : 1000, cat. no. 2983, CST), p-mTOR (1 : 1000, cat. no. 5536, CST), p53 (1 : 1000, cat. no. 2524, CST), p21 (1 : 1000, cat. no. ab109199, Abcam), gamma-H2AX (1 : 5000, cat. no. ab81299, Abcam), eNOS (1 : 1000, cat. no. 32027, CST), p62 (1 : 1000, cat. no. ab91526, Abcam), lc3B (1 : 1000, cat. no. 43566, CST), and β -actin (1 : 1000, cat. no. 20536-1-AP, Proteintech).

Techniques: Western Blot, Staining, Transmission Assay, Electron Microscopy, Confocal Microscopy, Quantitative RT-PCR