Journal: Oxidative Medicine and Cellular Longevity

Article Title: Melatonin Inhibits the Ferroptosis Pathway in Rat Bone Marrow Mesenchymal Stem Cells by Activating the PI3K/AKT/mTOR Signaling Axis to Attenuate Steroid-Induced Osteoporosis

doi: 10.1155/2022/8223737

Figure Lengend Snippet: MT reduces ferroptosis in DEX-induced BMSCs by activating the PI3K-AKT-mTOR pathway. (a–d) Western blot results for the expressions of PI3K-p85, p-AKT, AKT, p-mTOR, and mTOR were pretreated with various concentrations of MT for 24 h; DEX (10 −3 M) was then added for 24 h. (e) Images of immunofluorescence staining of PI3K in BMSCs. (f) Quantification of the fluorescence intensity of PI3K immunofluorescence positively stained cells. These studies were performed at least 3 biological replicates. Data represent mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.005 compared with control group. # P < 0.05, ## P < 0.01, ### P < 0.005 compared with DEX group.

Article Snippet: The primary antibodies included system xc − (1 : 2,000, orb100617, Biorbyt), ACSL4 (1 : 10,000, ab155282, Abcam), FSP1 (1 : 1,000, ab197896, Abcam), GPX4 (1 : 2,000, orb340797, Biorbyt), PI3K-p85 (1 : 1,000, orb621689, Biorbyt), AKT (1 : 1,000, orb99431, Biorbyt), p-ATK (1 : 1,000, orb344403, Biorbyt), mTOR (1 : 2,000, orb99435, Biorbyt), and p-mTOR (1 : 1,000, orb315804, Biorbyt) antibodies.

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Melatonin Inhibits the Ferroptosis Pathway in Rat Bone Marrow Mesenchymal Stem Cells by Activating the PI3K/AKT/mTOR Signaling Axis to Attenuate Steroid-Induced Osteoporosis

doi: 10.1155/2022/8223737

Figure Lengend Snippet: The positive regulatory effect of MT was significantly weakened with the use of PI3K inhibitors. (a–d) Western blot results for the expressions of PI3K-p85, p-AKT, AKT, p-mTOR, and mTOR were pretreated with GDC-0941 and MT for 24 h; DEX (10 −3 M) was then added for 24 h. (e) Images of immunofluorescence staining of PI3K in BMSCs. (f) Quantification of the fluorescence intensity of PI3K immunofluorescence positively stained cells. (g) Annexin V-mCherry/SYTOX Green detection kit was used to detect cell death. (h) Quantitative analysis of the percentage of SYTOX green-positive cells in (g). (i) ROS staining was performed to test the level of oxidative stress in DEX + MT group and DEX + MT + GDC0941 group. (j) Quantitative analysis of the number of ROS-positive cells per field in (i). These studies were performed at least 3 biological replicates. Data represent mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.005 compared with DEX + MT group.

Article Snippet: The primary antibodies included system xc − (1 : 2,000, orb100617, Biorbyt), ACSL4 (1 : 10,000, ab155282, Abcam), FSP1 (1 : 1,000, ab197896, Abcam), GPX4 (1 : 2,000, orb340797, Biorbyt), PI3K-p85 (1 : 1,000, orb621689, Biorbyt), AKT (1 : 1,000, orb99431, Biorbyt), p-ATK (1 : 1,000, orb344403, Biorbyt), mTOR (1 : 2,000, orb99435, Biorbyt), and p-mTOR (1 : 1,000, orb315804, Biorbyt) antibodies.

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Melatonin Inhibits the Ferroptosis Pathway in Rat Bone Marrow Mesenchymal Stem Cells by Activating the PI3K/AKT/mTOR Signaling Axis to Attenuate Steroid-Induced Osteoporosis

doi: 10.1155/2022/8223737

Figure Lengend Snippet: Schematic illustration of MT-mediated protection from GC-induced ferroptosis. Melatonin inhibits the ferroptosis pathway in rat bone marrow mesenchymal stem cells by activating the PI3K/AKT/mTOR signaling axis to attenuate steroid-induced osteoporosis.

Article Snippet: The primary antibodies included system xc − (1 : 2,000, orb100617, Biorbyt), ACSL4 (1 : 10,000, ab155282, Abcam), FSP1 (1 : 1,000, ab197896, Abcam), GPX4 (1 : 2,000, orb340797, Biorbyt), PI3K-p85 (1 : 1,000, orb621689, Biorbyt), AKT (1 : 1,000, orb99431, Biorbyt), p-ATK (1 : 1,000, orb344403, Biorbyt), mTOR (1 : 2,000, orb99435, Biorbyt), and p-mTOR (1 : 1,000, orb315804, Biorbyt) antibodies.

Techniques:

Journal: Cell Death & Disease

Article Title: Microglia as modulators of exosomal alpha-synuclein transmission

doi: 10.1038/s41419-019-1404-9

Figure Lengend Snippet: Whole-cell lysates were analyzed by western blot. a Protein levels of LC3 ( n = 5, ** p = 0.0082), P62/SQSTM1 ( n = 5, **** p < 0.0001), Beclin ( n = 5, * p = 0.0101), phospho-AKT ( n = 5, *** p = 0.0004), total-AKT, phospho-mTOR ( n = 4, * p = 0.0183), and β-actin were shown in indicated cells. b Whole-cell lysates were immunoblotted against α-syn (oligomeric) ( n = 5, ** p = 0.0078); monomeric ( n = 4, * p = 0.0113) and β-actin for control. c The triple-stained image of P62/SQSTM1 (gray) with IBA1 (green) and DAPI (blue) in the SNpc

Article Snippet: The following primary antibodies were utilized: rabbit antibody to IBA1 (1:1000, 10904-1-AP, Proteintech), mouse antibody to tsg 101 (1:500, sc-7964, Santa cruz biotechnology), mouse antibody to CD63 (1:1000, ab59479, Abcam), rabbit antibody to Calnexin (1:1000, 10427-2-AP, Proteintech), rabbit antibody to α-syn (1:1000, ab138501, ab52168, Abcam), rabbit antibody to Oligomer (1:1000, AHB0052, ThermoFisher scientific), rabbit antibody to TH (1:1000, 25859-1-AP, Proteintech), rabbit antibody to LC3 (1:1000, 14600-1-AP, Proteintech), rabbit antibody to SQSTM1/P62 (1:1000, ab91526, Abcam), rabbit antibody to Beclin1 (1:1000, 11306-1-AP, Proteintech), rabbit antibody to phosphor-AKT (1:800, AP0655, Abclonal), rabbit antibody to AKT (1:800, A11030, Abclonal), rabbit antibody to phosphor-mTOR (1:400, BM4840, Boster), mouse antibody to α-syn (1:1000, synuclein Ab-2, Thermo Scientific), and rabbit antibody to p-syn (1:500, GTX50222, Genetex).

Techniques: Western Blot, Control, Staining