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: The FASEB Journal

Article Title: Dietary polyphenols promote resilience against sleep deprivation‐induced cognitive impairment by activating protein translation

doi: 10.1096/fj.201800030r

Figure Lengend Snippet: Figure 3. BDPP activates the mTORC1 signaling pathway in the hippocampus. Hippocampal extracts were collected from NSD vehicle-treated mice, SD vehicle-treated mice, and SD mice treated with BDPP that were trained for OL test and sleep deprived for 5 h. A–D) ELISA-based quantification of protein abundance of mTOR (1-way ANOVA F2,23 = 1.829, P = 0.18; A), p70S6K (1- way ANOVA F2,16 = 0.977, P = 0.40; B), RPS6 (1-way ANOVA F2,21 = 0.387, P = 0.68; C), and 4E-BP1 (1-way ANOVA F2,21 = 0.457, P = 0.63; D). E–H) Quantification of phosphorylation abundance was calculated as the ratio of phosphorylated to total mTOR (p- mTOR, Ser2481, 1-way ANOVA, F2,23 = 10.15, P = 0.0008, Bonferroni post hoc test; E). ***P , 0.0005 NSD vs. SD and SD vs. SD + BDPP; p70S6K (p-p70S6K, Thr421/Ser424, 1-way ANOVA, F2,16 = 3.997, P = 0.04, Bonferroni post hoc test; F). *P , 0.05 NSD vs. SD; RPS6 (p-RPS6, Ser235/236, 1-way ANOVA, F2,21 = 2.487, P = 0.10, Bonferroni post hoc test; G). *P , 0.05 SD vs. SD + BDPP. 4E- BP1 (p-4E-BP1, Thr36, 1-way ANOVA, F2,21 = 2.821, P = 0.08, Bonferroni post hoc test; H). *P , 0.05 SD vs. SD + BDPP. Data are presented as fold change of means 6 SEM; n = 6–8 mice per condition. I–L) Luminex xMAP multiplexed immunoassays based quantification of total protein abundance of AKT (1-way ANOVA F2,11 = 0.244, P = 0.78; I), ERK1/2 (1-way ANOVA F2,11 = 0.177, P = 0.84; J), and quantification of phosphorylation abundance calculated as the ratio of phosphorylated to total of AKT (p-AKT, Ser473, 1-way ANOVA, F2,11 = 0.035, P = 0.96; K), and ERK1/2 (p-ERK1/2, Thr185/Tyr187, 1-way ANOVA, F2,11 = 3.287, P = 0.08; Bonferroni post hoc test; L). *P , 0.05 NSD vs. SD. Data are presented as fold change of means 6 SEM; n = 4 mice per condition. M) BDPP enhances protein translation and memory consolidation via activation of mTORC1 and downstream signaling pathway.

Article Snippet: Density of the signal was normalized to b-actin (1:1000; Cell Signaling Technology) The concentration of total and phosphorylated mTOR, p70S6K, RPS6, and 4E-BP1 in hippocampal or cell protein lysate was determined using commercially available ELISA kits (mTOR: PEL-mTOR-S2448-1; p70S6K: PEL-p70S6K-T421-1; RPS6: PEL-RPS6-S235-T-1; 4E-BP1: PEL-4EBP1-T36-T-1; RayBiotech), according to the manufacturer’s recommended procedures.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative Proteomics, Phospho-proteomics, Luminex, Activation Assay

Journal: The FASEB Journal

Article Title: Dietary polyphenols promote resilience against sleep deprivation‐induced cognitive impairment by activating protein translation

doi: 10.1096/fj.201800030r

Figure Lengend Snippet: Figure 4. Specific phenolic metabolites promote mTORC1 signaling pathway activation. Primary neuronal cultures were treated with phenolic metabolites, either alone or in combination, to examine their effect on the mTORC1 signaling pathway. Polyphenols and doses were chosen on the basis of the slope direction (positive/negative) of dose- response curves and calculated EC50 (Supplemental Table 5). For promoting effect, cultures were treated with CYA (0.1 nM), HPP (0.15 mM), or a combination of CYA (0.1 nM) +HPP (0.15 mM). For control, effect cultures were treated with DEL (0.1 nM), HBA (0.03 mM), or a combination of DEL (0.1 nM) + HBA (0.03 mM). Control cells were treated with DMSO. After 24 h of treatment, cells were washed once with ice-cold PBS and protein was extracted. ELISA-based assay was used for quantification of fold change in total protein abundance of mTOR (A), p70S6K (B), RPS6 (C), 4E-BP1 (D), and quantification of fold of change in phosphorylated protein abundance calculated as ratio of phosphorylated to total protein of mTOR (p-mTOR, Ser2481; E), p70S6K (p-p70S6K, Thr421/Ser424; F), RPS6 (p-RPS6, Ser235/236; G), and 4E-BP1 (p-4E- BP1, Thr36; H). Data are presented as means 6 SEM; n = 4–7 samples/condition. *P , 0.05, unpaired, 2-tailed Student’s t test, treatment vs. Ctrl.

Article Snippet: Density of the signal was normalized to b-actin (1:1000; Cell Signaling Technology) The concentration of total and phosphorylated mTOR, p70S6K, RPS6, and 4E-BP1 in hippocampal or cell protein lysate was determined using commercially available ELISA kits (mTOR: PEL-mTOR-S2448-1; p70S6K: PEL-p70S6K-T421-1; RPS6: PEL-RPS6-S235-T-1; 4E-BP1: PEL-4EBP1-T36-T-1; RayBiotech), according to the manufacturer’s recommended procedures.

Techniques: Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics