Journal: Molecular Medicine Reports

Article Title: Effect of Salvia miltiorrhiza and ligustrazine injection on myocardial ischemia/reperfusion and hypoxia/reoxygenation injury

doi: 10.3892/mmr.2016.5822

Figure Lengend Snippet: Effect of SLI on Akt and eNOS levels. The protein expression levels of Akt and eNOS was determined by western blot analysis following H/R injury. Pretreatment of 0.1 uM SLI improved the ratio of (A) p-Akt/Akt and (B) p-eNOS/eNOS. The Akt inhibitor, LY294002, significantly inhibited the ratio of p-Akt/Akt. Data are presented as the mean ± standard deviation ( ## P<0.05 vs. control group; *P<0.01 vs. SLI group; **P<0.01 vs. H/R group). H/R, hypoxia/reoxygenation; SLI, Salvia miltiorrhiza and ligustrazine injection; p-Akt, phosphorylated Akt serine/threonine kinase; p-eNOS, phosphorylated-endothelial nitric oxide synthase; veh, vehicle; con, control.

Article Snippet: Following blocking, the membranes were incubated with the primary antibodies against rabbit IgG p-eNOS/eNOS (1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), rabbit IgG p-Akt/Akt (1:1,000; BD Biosciences), Bcl-2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), Bax (1:2,000; Cell Signaling Technology, Inc.) and caspase-3 (1:3,000; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Expressing, Western Blot, Standard Deviation, Injection

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Antihypertrophic effects of OT in vitro . Neonatal rat cardiomyocytes stimulated with ISO for 24 h in the presence or absence of OT. (A) The cell morphology was evaluated by H&E staining. (B) Statistical results of measurement of cell surface areas. (C,D) Effects of OT on the protein expressions of BNP and β-MHC. (E) Effects of OT on the expression of lncRNA GAS5. (F) Effects of OT on the expression of miR-375-3p. (G,H) Effects of OT on the mRNA and protein expressions of KLF4. (I) Effects of OT on the p-PI3K/PI3K ratio. (J) Effects of OT on the p-AKT1/AKT1 ratio. (K) Western blot images of BNP, β-MHC, KLF4, p-PI3K, PI3K, AKT1, p-AKT1, and β-actin levels. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST).

Techniques: In Vitro, Staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Over-expression of miR-375-3p blunted anti-hypertrophic effects of oxytocin via KLF4 and modulated the PI3K/AKT signaling pathway. (A) Expression of miR-375-3p in cardiomyocytes following transfection of miR-375-3p mimics and miR-375-3p mimics NC. (B) Expression of miR-375-3p was detected by qRT-PCR. (C,D) Expressions of BNP and β-MHC proteins were measured by Western blot analysis. (E) Expression of KLF4 mRNA was detected by qRT-PCR. (F) Expression of KLF4 protein was measured by Western blot analysis. (G) p-PI3K/PI3K ratio. (H) p-AKT1/AKT1 ratio. (I) Statistical results of measurement of cell surface areas. (J) Representative western blot images of KLF4, PI3K, p-PI3K, AKT1, p-AKT1, BNP, β-MHC and GAPDH levels. (K) Cardiomyocyte surface areas were measured by immunofluorescent staining. Scale bars represent 20 µm. Images were captured at ×400 magnification. (a) ISO+OT+miR-375-3p mimics NC group. (b) ISO+OT+miR-375-3p mimics group. (c) ISO+OT+miR-375-3p mimics+pcDNA-NC group. (d) ISO+OT+miR-375-3p mimics+pcDNA-KLF4 group. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST).

Techniques: Over Expression, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: knock-down of KLF4 blunted anti-hypertrophic effects of oxytocin via PI3K/AKT pathway. (A–C) Detection of relative shRNA-KLF4 interference effects. The changes in KLF4 mRNA and protein levels were detected by qRT-PCR and Western blotting after transfection of shRNA-KLF4 #1, shRNA-KLF4 #2, shRNA-KLF4 #3 in primary cardiomyocytes. (D) Expression of KLF4 mRNA was determined by qRT-PCR. (E) Expression of KLF4 protein was measured by Western blot analysis. (F,G) Expressions of BNP, β-MHC proteins were measured by Western blot analysis. (H) p-PI3K/PI3K ratio. (I) p-AKT1/AKT1 ratio. (J) Statistical results of measurement of cell surface areas. (K) Cardiomyocyte surface areas were measured by immunofluorescent staining. Scale bars represent 20 µm. Images were captured at ×400 magnification. (L) Western blot images of KLF4, PI3K, p-PI3K, AKT1, p-AKT1, BNP, β-MHC and GAPDH levels. (a) ISO+OT+shRNA-NC group. (b) ISO+OT+shRNA-KLF4 group. (c) ISO+OT+shRNA-KLF4+LY194002 group. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST).

Techniques: shRNA, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Staining

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Oligonucleotide sequences used in this study.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Primers used for plasmids construction.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: miR-146b-3p targets AKT1 and MDFIC and downregulates PI3K/AKT pathway activity. ( a ) Relative mRNA expression of AKT1 and MDFIC in QM-7 cells after overexpression or inhibition of miR-146b-3p. ( b ) The phosphorylation levels of AKT and AKT1 and the protein expression of AKT1 after transfecting miR-146b-3p mimic or inhibitor in CPMs. ( c ) Gray value analysis of protein bands in (b). ( d ) Dual-luciferase report assay performed after co-transfecting the wild type or mutant 3′UTR of AKT1 with miR-146b-3p mimic or mimic NC in DF-1 cells. ( e ) Dual-luciferase report assay performed after co-transfecting the wild type or mutant 3′UTR of MDFIC with miR-146b-3p mimic or mimic NC in DF-1 cells. ( f ) Relative mRNA expression of the cell differentiation-related genes after co-transfection. ( g ) Relative mRNA expression of the cell apoptosis-related genes after co-transfection. ( h ) Cell cycle analysis of QM-7 cells after co-transfection. Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Activity Assay, Expressing, Over Expression, Inhibition, Luciferase, Mutagenesis, Cell Differentiation, Cotransfection, Cell Cycle Assay, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: AKT1 and MDFIC promote myoblast proliferation. ( a ) Cell cycle analysis of CPMs after overexpression or inhibition of AKT1 . ( b ) Cell cycle analysis of CPMs after overexpression or inhibition of MDFIC . ( c ) Relative mRNA expression of the cell cycle-related genes after overexpression or inhibition of AKT1 in CPMs. ( d ) Relative mRNA expression of the cell cycle-related genes after overexpression or inhibition of MDFIC in CPMs. ( e ) EdU staining of CPMs after overexpression or inhibition of AKT1 . ( f ) The fold change of proliferation rates of CPMs with pcDNA3.1- AKT1 or si- AKT1 . ( g ) EdU staining of CPMs after overexpression or inhibition of MDFIC . ( h ) The fold change of proliferation rates of CPMs with pcDNA3.1- MDFIC or si- MDFIC . Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Cell Cycle Assay, Over Expression, Inhibition, Expressing, Staining, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Both AKT1 and MDFIC promote myoblast differentiation, while suppress myoblast apoptosis. ( a ) Relative expression of AKT1 in Xinghua chicken leg muscle from E11 to E18. ( b ) Relative expression of MDFIC in Xinghua chicken leg muscle from E11 to E18. ( c ) Relative mRNA expression of AKT1 during CPMs induced differentiation. ( d ) Relative mRNA expression of MDFIC during CPMs-induced differentiation. ( e , f ) Relative mRNA expression of the cell differentiation-related genes after overexpression or inhibition of AKT1 or MDFIC in CPMs. ( g , i ) Immunofluorescence of MyHC after overexpression or knockdown of AKT1 and MDFIC in CPMs. ( h , j ) Comparison of the area of myotubes described in ( g , i ). Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Expressing, Cell Differentiation, Over Expression, Inhibition, Immunofluorescence, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: AKT1 shows an inhibitory effect on myoblast apoptosis and so does MDFIC . ( a , b ) Flow cytometry analysis of Annexin V-FITC and PI dual staining detecting the apoptosis of CPMs after transfection of pcDNA3.1- AKT1 or si- AKT1 . ( c ) Relative mRNA expression of the cell apoptosis-related genes after transfection of pcDNA3.1- AKT1 or si- AKT1 in CPMs. ( d ) Relative mRNA expression of the cell apoptosis-related genes after transfection of pcDNA3.1- MDFIC or si- MDFIC in CPMs. ( e , f ) The protein levels of MyHC, cleaved-caspase 8, and cleaved-caspase 9 after the overexpression or silence of AKT1 and MDFIC in CPMs. ( g , h ) Gray value analysis of protein bands in ( e , f ). Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Flow Cytometry, Staining, Transfection, Expressing, Over Expression, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Model of miR-146b-3p mediated regulatory mechanism in myoblast proliferation, differentiation, and apoptosis. In simple terms, miR-146b-3p downregulates the expression of AKT1 and MDFIC by targeting the 3′UTR of their mRNA. Both the phosphorylation of AKT1 and AKT can trigger the activation of PI3K/AKT pathway, thereby promoting the expression of cell cycle-related genes and myoblast differentiation-related genes and suppress cell apoptosis-related genes, which in turn facilitate cell proliferation and differentiation and inhibit apoptosis. In addition, the expression of MDFIC can also lead to the same effect.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Expressing, Activation Assay