Journal: Frontiers in Neuroscience

Article Title: High-Frequency Repetitive Transcranial Magnetic Stimulation Mediates Autophagy Flux in Human Bone Mesenchymal Stromal Cells via NMDA Receptor–Ca 2+ –Extracellular Signal-Regulated Kinase–Mammalian Target of Rapamycin Signaling

doi: 10.3389/fnins.2019.01225

Figure Lengend Snippet: Repetitive transcranial magnetic stimulation (rTMS) mediates autophagy via mTOR. (A) p-mTOR and mTOR assessed by western blotting. (B) Quantification of western blotting for p-mTOR/mTOR. Data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison test. ∗ P < 0.05, n = 3. Error bars = SD. The value of p-mTOR/mTOR in the 0.5 T group was 0.301 ± 0.223. (C) p-mTOR and mTOR assessed by western blotting after normal group cotreatment with 3BDO. (D) Quantification of western blotting for p-mTOR/mTOR. Data were analyzed with a Student’s t -test. ∗ P < 0.05, n = 3. Error bars = SD. The value of p-mTOR/mTOR in the MK801 group was 2.200 ± 0.283. (E) After cotreatment with 3BDO, the expression of p-mTOR, mTOR, LC3, and p62 was assessed by western blotting. (F–H) Quantification of western blotting for p-mTOR/mTOR, LC3-II/I, and p62. Data were analyzed with a Student’s t -test. ∗ P < 0.05, ∗∗ P < 0.01, n = 3. Error bars = SD. The values of p-mTOR/mTOR, p62, and LC3-II/I in the 0.5 T + 3BDO group were 1.950 ± 0.212, 1.825 ± 0.106, and 0.790 ± 0.057, respectively. mTOR, mammalian target of rapamycin.

Article Snippet: All reagents are listed as follows: phosphorylated-AKT (p-AKT) (4060, Cell Signaling), AKT (4691, Cell Signaling), phosphorylated extracellular signal-regulated kinase (p-ERK) (50011, Abcam), ERK (184699, Abcam), phosphorylated epidermal growth factor receptor (p-EGFR) (40815, Abcam), EGFR (52894, Abcam), NMDA receptor (NMDAR1) (17345, Abcam), p62 (56416, Abcam), LC3 (192890, Abcam), phosphorylated mammalian target of rapamycin (p-mTOR) (5536, Cell Signaling), mTOR (2983, Cell Signaling), phosphorylated protein kinase A (p-PKA) (32390, Abcam), PKA (75993, Abcam), Oct4 (2750, Cell Signaling), Nanog (8822, Cell Signaling), Sox2 (3579, Cell Signaling), GAPDH (8245, Abcam), dizocilpine maleate (MK801) (15084, MCE), 1,4-diamino-2,3-dicyano-1,4-bis( o -aminophenylmercapto)butadiene (U0126) (1901, Beyotime), 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3 H )-one (3BDO) (8317, Selleck), Fluo-4/AM (1060, Beyotime), horseradish peroxidase (HRP)-mouse (931, Sigma), HRP-rabbit (934, Sigma), Alexa Fluor 488 (150077, Abcam), Alexa Fluor Cy3 (97035, Abcam), and Cell Counting Kit-8 (CCK-8) (0037, Beyotime).

Techniques: Western Blot, Comparison, Expressing

Journal: Frontiers in Neuroscience

Article Title: High-Frequency Repetitive Transcranial Magnetic Stimulation Mediates Autophagy Flux in Human Bone Mesenchymal Stromal Cells via NMDA Receptor–Ca 2+ –Extracellular Signal-Regulated Kinase–Mammalian Target of Rapamycin Signaling

doi: 10.3389/fnins.2019.01225

Figure Lengend Snippet: Effects of MK801 on NMDAR1 after incubation with rTMS in bone mesenchymal stromal cells (BMSCs). (A) NMDAR1 assessed by western blotting after normal group cotreatment with MK801. (B) Quantification of western blotting for NMDAR1. Data were analyzed with a Student’s t -test. ∗∗ P < 0.01, n = 3. Error bars = SD. The value of NMDAR1 in the MK801 group was 0.130 ± 0.042. (C) Detection of Ca 2+ levels after normal group cotreatment with MK801. (D) Quantitative fluorescence intensity of Ca 2+ levels. Data were analyzed with a Student’s t -test. ∗ P < 0.05, n = 3. Error bars = SD. The value of Ca 2+ in the MK801 group was 0.650 ± 0.071. (E) Cells were pretreated with MK801 (10 μM) for 1 h and incubated with rTMS. NMDAR1, ERK, p-ERK, p-mTOR, mTOR, LC3, and p62 were assessed by western blotting. (F–J) Quantification of western blotting for NMDAR1, p-ERK/ERK, p-mTOR/mTOR, LC3-II/I, and p62. Data were analyzed with a Student’s t -test. ∗ P < 0.05, ∗∗ P < 0.01, n = 3. Error bars = SD. The values of NMDAR1, p-ERK/ERK, p-mTOR/mTOR, p62, and LC3-II/I in the 0.5 T + MK801 group were 0.200 ± 0.141, 0.450 ± 0.071, 1.735 ± 0.092, 1.715 ± 0.177, and 0.770 ± 0.042, respectively. (K) Detection of intracellular Ca 2+ levels after cotreatment with MK801. (L) Quantitative fluorescence intensity of Ca 2+ levels assessed by Fura-4/AM. Data were analyzed with a Student’s t -test. ∗ P < 0.05, n = 3. Error bars = SD. The value of Ca 2+ in the 0.5 T + MK801 group was 0.775 ± 0.035. ERK, extracellular signal-regulated kinase; mTOR, mammalian target of rapamycin.

Article Snippet: All reagents are listed as follows: phosphorylated-AKT (p-AKT) (4060, Cell Signaling), AKT (4691, Cell Signaling), phosphorylated extracellular signal-regulated kinase (p-ERK) (50011, Abcam), ERK (184699, Abcam), phosphorylated epidermal growth factor receptor (p-EGFR) (40815, Abcam), EGFR (52894, Abcam), NMDA receptor (NMDAR1) (17345, Abcam), p62 (56416, Abcam), LC3 (192890, Abcam), phosphorylated mammalian target of rapamycin (p-mTOR) (5536, Cell Signaling), mTOR (2983, Cell Signaling), phosphorylated protein kinase A (p-PKA) (32390, Abcam), PKA (75993, Abcam), Oct4 (2750, Cell Signaling), Nanog (8822, Cell Signaling), Sox2 (3579, Cell Signaling), GAPDH (8245, Abcam), dizocilpine maleate (MK801) (15084, MCE), 1,4-diamino-2,3-dicyano-1,4-bis( o -aminophenylmercapto)butadiene (U0126) (1901, Beyotime), 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3 H )-one (3BDO) (8317, Selleck), Fluo-4/AM (1060, Beyotime), horseradish peroxidase (HRP)-mouse (931, Sigma), HRP-rabbit (934, Sigma), Alexa Fluor 488 (150077, Abcam), Alexa Fluor Cy3 (97035, Abcam), and Cell Counting Kit-8 (CCK-8) (0037, Beyotime).

Techniques: Incubation, Western Blot, Fluorescence