Journal: International journal of molecular medicine

Article Title: Nicorandil protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis.

doi: 10.3892/ijmm.2015.2229

Figure Lengend Snippet: Figure 3. Nicorandil activates the PI3K/Akt signaling pathway under H/SD conditions. MSCs were treated with nicorandil (100 µM) for the indicated periods of under H/SD conditions for 9 h. (A and C) The levels of p-Akt was upregulated by nicorandil in a time-dependent manner, peaking at 90 min. Data are presented as the means ± SD of 3 separate experiments. *P<0.05, compared with the normal group; ▲P<0.05, compared with the group treated with nicorandil for 90 min. (B and D) The inhibition of PI3K with LY294002 triggered p-Akt inactivation. (E and F) Nicorandil induced a significant decrease in the apoptotic rate of MSCs under H/SD conditions, which was reversed by LY294002. Data are presented as the means ± SD of 3 separate experiments. *P<0.05, compared with the normal group; ▲P<0.05, compared with the 100 µM nicorandil-treated group. PI3K, phosphoinositide 3-kinase; MSCs, mesenchymal stem cells; H/SD, hypoxia/serum deprivation; Nico, nicorandil; p-Akt, phosphorylated Akt (Ser473); t-Akt, total Akt; NAC, N-acetyl-L-cysteine.

Article Snippet: The protein band was transferred onto polyvinylidene difluoride (PVDF) membranes blocked with 8% fat-free milk in Tris-buffered saline (TBS) with 0.5% Tween-20 for 60 min at 37 ̊C, followed by treatment with the following primary antibodies at 4 ̊C overnight: rabbit monoclonal against Akt (cst-4691s), phosphorylated Akt [p-Akt (Ser473); cst-4060s], caspase-3 (cst-9662s), Bax (cst-2772s), Bcl-2 (cst-2876s) and cytochrome c (cst-4272s) (all from Cell Signaling Technology) and mouse polyclonal antibody against β-actin (TA-09; Zhongshan Golden Bridge Biotechnology, Beijing, China).

Techniques: Inhibition

Journal: International journal of molecular sciences

Article Title: Improved Wound Healing and Skin Regeneration Ability of 3,2'-Dihydroxyflavone-Treated Mesenchymal Stem Cell-Derived Extracellular Vesicles.

doi: 10.3390/ijms24086964

Figure Lengend Snippet: Figure 2. Characterization of 3,2′-DHF-treated WJ-MSCs and EVs. (A) The proliferation rate of WJ- MSC treated with various concentrations of 3,2′-DHF. WJ-MSCs have the best proliferation rate with 1–2 µM 3,2′-DHF. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test: ** p < 0.05, **** p < 0.0001. (B) Protein expression of MEK/ERK signaling proteins in WJ-MSCs with and without 3,2′-DHF treatment. The bar graph shows the protein expression of AKT and MEK in 3,2′-DHF-treated and untreated WJ-MSCs. (C) Flow cytometry analysis to confirm the characteristics of WJ-MSCs with or without 3,2′-DHF. The expression of the positive markers CD73, CD90, and CD105, and the negative markers CD34 and CD45, was assessed. (D) The relative EV production amounts of Cont-EV and Fla-EV were measured using nanoparticle tracking analysis (NTA). Statistical analysis was determined using two-way ANOVA: **** p < 0.0001. (E) Transmission electron microscopy (TEM) images of Cont-EV and Fla-EV. Scale bar: 100 nm. The size of the EVs was determined with NTA. (F) Characterization of EV-positive markers CD9 and CD63, and negative markers calnexin and GM130, in Cont-EV and Fla-EV using Western blotting. (G) Representative plot of EV-surface markers CD63 and CD81 in Cont-EV and Fla-EV via flow cytometry analysis.

Article Snippet: The following antibodies were diluted in 1× blocking buffer (TLP-115.1G, Translab): CD9 (ab263023, Abcam, Cambridge, UK), CD63 (ab109201, Abcam), calnexin (2679, Cell Signaling Technology, Danvers, MA, USA), GM130 (12480, Santa Cruz Biotechnology), p-AKT (sc-293125, Santa Cruz Biotechnology), AKT (CSB-PA000855, Cusabio, Houston, TX, USA), p-ERK (CSB-PA000749, Cusabio), ERK (B7074, Tebu-bio, Le Perray, Ile-de-France, France), β-actin (sc-47778, Santa Cruz Biotechnology), HRP-linked anti-rabbit IgG (7074, Santa Cruz Biotechnology), and HRP-linked anti-mouse IgG (7076, Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Transmission Assay, Electron Microscopy, Western Blot, Cytometry

Journal: Clinical Medicine Insights. Oncology

Article Title: MicroRNA-19b Downregulates NR3C1 and Enhances Oxaliplatin Chemoresistance in Colon Cancer via the PI3K/AKT/mTOR Pathway

doi: 10.1177/11795549211012666

Figure Lengend Snippet: The miR-19b inhibition and NR3C1 restoration cause decreased cell viability and apoptosis via the PI3K/AKT/mTOR pathway. (A) The expression of AKT, NR3C1, p-AKT, mTOR, p-mTOR, GSK-3β, and p-GSK-3β was detected by Western blotting. After miR-19b inhibition, the levels of p-AKT (Thr308), p-AKT (Ser473), p-mTOR (Ser2448), and p-GSK-3β (Ser9) were significantly decreased. (B) Cell viability suppression; (C) increased inhibition rate; and (D) apoptotic rate, caused by miR-19b inhibitor partially reversed by PI3K/AKT and mTOR pathway activators. (E) Cell viability suppression; (F) increased inhibition rate; and (G) apoptotic rate, caused by NR3C1 overexpression partially reversed by PI3K/AKT and mTOR pathway activators. * P < .05, when compared with # .

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) and incubated respectively with NR3C1 antibody (Origene, MD; 1:800), AKT (Invitrogen, New York, NY; 1:1000), p-AKT (Thr308) (Invitrogen, New York, NY; 1:800), p-AKT (Ser473) (Invitrogen; 1:500), mTOR (Abcam, MA; 1:800), p-mTOR (Abcam; 1:1000), GSK-3β (Abcam; 1:800), p-GSK-3β (Abcam; 1:1000), and β-Actin (Santa Cruz, CA; 1:1000).

Techniques: Inhibition, Expressing, Western Blot, Over Expression