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

Journal: Journal of Nutrition and Metabolism

Article Title: Undaria pinnatifida (Wakame) Intake Ameliorates High-Fat Diet-Induced Glucose Intolerance via Promoting GLUT4 Expression and Membrane Translocation in Muscle

doi: 10.1155/2023/9774157

Figure Lengend Snippet: Effect of wakame components on AKT and AMPK phosphorylation and GLUT4 levels in C2C12 cells. Differentiated C2C12 cells were treated with PBS, DMSO, metformin (1 mM), fucoidan (1 mg/mL), ethanol extract (50 μ g/mL), hexane extract (50 μ g/mL), fucoxanthinol (1 μ g/mL), or wakame peptide (100 μ g/mL) for 24 h before lysis. The lysates were analyzed with Western blot using (a) GLUT4, (b) P-AKT (s473) and T-AKT, and (c) P-AMPK (T172) and T-AMPK antibodies and quantified. The level of GLUT4 was normalized by that of GAPDH. The intensity of P-AKT and P-AMPK bands was normalized by that of their respective total protein bands. The data are presented as mean ± SD ( n = 4). The data were analyzed with one-way ANOVA combined with the Tukey test. ∗ p < 0.05, compared to PBS; # p < 0.05, compared to DMSO.

Article Snippet: The phosphor-specific antibodies, P-AKT (S473, 4051S; Cell Signaling Technology) and P-AMPK (T172, 2531S; Cell Signaling Technology), were used at 1 : 1000 dilution.

Techniques: Lysis, Western Blot

Journal: Journal of Nutrition and Metabolism

Article Title: Undaria pinnatifida (Wakame) Intake Ameliorates High-Fat Diet-Induced Glucose Intolerance via Promoting GLUT4 Expression and Membrane Translocation in Muscle

doi: 10.1155/2023/9774157

Figure Lengend Snippet: Wakame's effect on level and phosphorylation of insulin signaling molecules and GLUT4 in skeletal muscle. The skeletal muscle cells were collected via dissection from the ND, ND + W, HFD, and HFD + W mice. The cell lysates were analyzed by Western blotting with (a) GLUT4, (b) P-AKT (s473) and T-AKT, and (c) P-AMPK (T172) and T-AMPK antibodies. The level of GLUT4 was normalized to that of GAPDH. The level of P-AKT and T-AMPK was normalized to that of their respective total protein. The data were presented as mean ± SD ( n = 4-5) and analyzed with one-way ANOVA combined with the Tukey test. ∗ p < 0.05.

Article Snippet: The phosphor-specific antibodies, P-AKT (S473, 4051S; Cell Signaling Technology) and P-AMPK (T172, 2531S; Cell Signaling Technology), were used at 1 : 1000 dilution.

Techniques: Dissection, Western Blot