Journal: PeerJ

Article Title: 7,8-Dihydroxyflavone protects neurons against oxygen-glucose deprivation induced apoptosis and activates the TrkB/Akt pathway.

doi: 10.7717/peerj.12886

Figure Lengend Snippet: Figure 1 Determination of a suitable concentration of 7,8-DHF and Flow Cytometric Analysis. (A) The cell viability was assessed using a cell counting kit-8 (CCK-8) assay compared with the control group. The cell viability was significantly increased in the OGD/R+7,8-DHF groups as compared with that in the OGD/R group and peaked in the 0.5 µmol OGD/R+7,8-DHF group. *p < 0.05. vs the control group; # p < 0.05 vs OGD/R group; □p < 0.05 vs 0.25 µmol 7,8-DHF+OGD/R group; ■p < 0.05 vs 0.75 µmol OGD/R+7,8-DHF group; ⃝p < 0.05 vs 1 µmol OGD/R+7,8-DHF group. (B–C) Neuronal apoptosis is analyzed by Flow Cytometric Analysis. Mean ± standard deviation (SD) values from 24 independent ex- periments are presented. ***p < 0.001. vs The Control group, ###p < 0.001. vs OGD/R group. Differences between the groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: 7,8-DHF (MedChemExpress, USA) and K252a (0.1 μmol) (Kim & Jin, 2020) (MedChemExpress, USA) were obtained as a powder and dissolved in dimethyl sulfoxide (DMSO) (one mmol with DMSO concentration adjusted o 0.1%), which were administrated after OGD/R for 0.5 h in an incubator under 95% air/5% CO2 at 37 ◦C till for 24 h Reperfusion.

Techniques: Concentration Assay, Cell Counting, CCK-8 Assay, Control, Standard Deviation

Journal: PeerJ

Article Title: 7,8-Dihydroxyflavone protects neurons against oxygen-glucose deprivation induced apoptosis and activates the TrkB/Akt pathway.

doi: 10.7717/peerj.12886

Figure Lengend Snippet: Figure 2 7,8-DHF inhibits OGD/R-induced cell apoptosis. (A) The TUNEL staining of neurons in the control, OGD/R, and OGD/R+7,8-DHF groups. (C) The semiquantitative analysis of TUNEL positive neurons in the three groups. (B) Western blotting of B-cell lymphoma 2 (Bcl-2), Cleaved-Caspase-3, and Bcl-2-associated X (Bax) expression in control, OGD/R, and OGD/R+7,8-DHF groups. The semiquanti- tative analysis of Bax (D), Bcl-2 (E), and cleaved-caspase 3 (F). Scale bar = 50 µm. Mean ± standard de- viation (SD) values are presented. *p < 0.05, **p < 0.01, ***p < 0.001. vs the control group.; #p < 0.05, # #p < 0.01, ###p < 0.001. vs OGD/R group. Differences between the groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: 7,8-DHF (MedChemExpress, USA) and K252a (0.1 μmol) (Kim & Jin, 2020) (MedChemExpress, USA) were obtained as a powder and dissolved in dimethyl sulfoxide (DMSO) (one mmol with DMSO concentration adjusted o 0.1%), which were administrated after OGD/R for 0.5 h in an incubator under 95% air/5% CO2 at 37 ◦C till for 24 h Reperfusion.

Techniques: TUNEL Assay, Staining, Control, Western Blot, Expressing

Journal: PeerJ

Article Title: 7,8-Dihydroxyflavone protects neurons against oxygen-glucose deprivation induced apoptosis and activates the TrkB/Akt pathway.

doi: 10.7717/peerj.12886

Figure Lengend Snippet: Figure 3 Effect of 7,8-DHF on expression of TrkB and Akt. (A) Western blotting of TrkB, pTrkB, Akt and pAkt in five groups. The semiquantitative analysis of pTrkB (B) and pAkt/Akt (C). *p < 0.05, **p < 0.01. vs the control group; Mean ± standard deviation (SD) values are presented. & p < 0.05, && p < 0.01. vs OGD/R group; #p < 0.05, ##p < 0.01. vs OGD/R+7,8-DHF group. Differences between the groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: 7,8-DHF (MedChemExpress, USA) and K252a (0.1 μmol) (Kim & Jin, 2020) (MedChemExpress, USA) were obtained as a powder and dissolved in dimethyl sulfoxide (DMSO) (one mmol with DMSO concentration adjusted o 0.1%), which were administrated after OGD/R for 0.5 h in an incubator under 95% air/5% CO2 at 37 ◦C till for 24 h Reperfusion.

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: PeerJ

Article Title: 7,8-Dihydroxyflavone protects neurons against oxygen-glucose deprivation induced apoptosis and activates the TrkB/Akt pathway.

doi: 10.7717/peerj.12886

Figure Lengend Snippet: Figure 4 Effect of 7, 8-DHF and K252a on expression of apoptosis related proteins. (A) Western blot- ting showing the expression of Bax, BCL-2 and Cleaved-Caspase3 in five groups. The semiquantitative analysis of Bax (C), Bcl-2 (B), and cleaved-caspase 3 (D). Mean ± standard deviation (SD) values are pre- sented. **p < 0.01, ***p < 0.001. vs the control group; &p < 0.05, &&p < 0.01, &&&p < 0.001. vs OGD/R group; ##p < 0.01, ###p < 0.001. vs OGD/R+7,8-DHF group. Differences between the groups were ana- lyzed using one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: 7,8-DHF (MedChemExpress, USA) and K252a (0.1 μmol) (Kim & Jin, 2020) (MedChemExpress, USA) were obtained as a powder and dissolved in dimethyl sulfoxide (DMSO) (one mmol with DMSO concentration adjusted o 0.1%), which were administrated after OGD/R for 0.5 h in an incubator under 95% air/5% CO2 at 37 ◦C till for 24 h Reperfusion.

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: PeerJ

Article Title: 7,8-Dihydroxyflavone protects neurons against oxygen-glucose deprivation induced apoptosis and activates the TrkB/Akt pathway.

doi: 10.7717/peerj.12886

Figure Lengend Snippet: Figure 5 The role of TrkB in neurons after 7,8-DHF treatment. (A) Western blotting showing a knock- down of TrkB. (B) The semiquantitative analysis of TrkB. (C) Effect of 7,8-DHF on related proteins with a knockdown of TrkB. The semiquantitative analysis of pTrkB (D), TrkB (E), pAkt/Akt (F), Bax (G), Bcl-2 (H) Cleavd-Caspase3 (I). Mean ± standard deviation (SD) values are presented. **p < 0.01, ***p < 0.001. vs the control group; &&p < 0.01, &&&p < 0.001. vs OGD/R group; #p < 0.05, ##p < 0.01, ###p < 0.001. vs OGD/R+7,8-DHF group. Differences between the groups were analyzed using one-way ANOVA fol- lowed by Tukey’s post hoc test.

Article Snippet: 7,8-DHF (MedChemExpress, USA) and K252a (0.1 μmol) (Kim & Jin, 2020) (MedChemExpress, USA) were obtained as a powder and dissolved in dimethyl sulfoxide (DMSO) (one mmol with DMSO concentration adjusted o 0.1%), which were administrated after OGD/R for 0.5 h in an incubator under 95% air/5% CO2 at 37 ◦C till for 24 h Reperfusion.

Techniques: Western Blot, Knockdown, Standard Deviation, Control

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Tissue-Type Plasminogen Activator Regulates the Neuronal Uptake of Glucose in the Ischemic Brain

doi: 10.1523/JNEUROSCI.1241-12.2012

Figure Lengend Snippet: A. Experimental design used to study the effect of tPA on neuronal survival. Letters denote time of treatment with tPA after exposure to oxygen-glucose deprivation (OGD) conditions. B – D. Mean cell survival (panels B & C) and release of LDH into the culture media (panel D) in Wt cerebral cortical neurons treated with 5 nM of proteolytically active (panels B & D) or inactive tPA (itPA; panels C & D), or 10 nM of plasmin (panel C), 5, or 30, or 60, or 120, or 180, or 360 minutes after exposure to 55 minutes of OGD conditions. n = 20 in B and 15 in C and D. * in B: p < 0.05 compared to neurons exposed to OGD conditions without subsequent treatment with tPA. * in C: p < 0.05 compared to neurons left untreated after exposure to OGD conditions. Ns: non-significant. * in D: p < 0.05 compared to cells exposed to OGD conditions without subsequent treatment. Lines denote SD. E. Mean cell survival in Wt cerebral cortical neurons exposed to 55 minutes of OGD conditions and treated 1 hour later with 5 nM of tPA, alone or in combination with either 10 µM of MK-801 or 60 nM of the receptor associated protein (RAP), or 100 nM of the tyrosine kinase receptor B (TrkB) inhibitor K-252a. n = 10. * p < 0.05 compared to neurons treated with tPA alone, or with a combination of tPA and K-252a. Lines denote SD. F. Mean volume of the ischemic lesion in Wt and Plg−/− mice treated one hour after tMCAO with saline solution (white bars) or rtPA 1 – 9 mg/Kg/IV (gray bars). n = 10 per group. * p < 0.05 compared to Wt mice treated with saline solution. ** p < 0.05 compared to mice treated with 1 mg/Kg/IV of rtPA. *** p < 0.05 compared to Plg−/− mice treated with saline solution. Bars depict mean volume of the ischemic lesion in mm3. Lines denote SD.

Article Snippet: Other reagents were human recombinant tissue-type plasminogen activator (Genentech Inc.), the phosphoinositide (PI) 3-kinase/Akt inhibitor Wortmannin, methanol, methyl salicylate and triphenyltetrazolium chloride (Sigma Aldrich), the 3-(4,5- Dimethylthiazol -2-yl)-2,5-di phenyl tetrazolium bromide (MTT) assay (ATCC), the LDH release assay (Roche), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801 (Tocris Bioscience), rapamycin and the TrkB inhibitor K-252a (Calbiochem), HIF-1α shRNA, scramble shRNA lentiviral particles, anti-GLUT3 antibodies and TRITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology), anti-HIF-1α antibodies (Abcam), antibodies against the p70S6 kinase (p70 S6K ) phosphorylated at Thr389 (Cell Signaling), heparin sodium (Abraxis Pharmaceutical Products), blue latex (Connecticut Valley Biological Supply), ApopTag Plus Fluorescein in Situ Apoptosis Detection Kit (Chemicon International), 4'-6-Diamidino-2-phenylindole (DAPI; Invitrogen), triphenyltetrazolium chloride (TTC; Sigma-Aldrich), 2- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Molecular Probes), and 18-Fluorodeoxyglucose (PETNET).

Techniques: Saline