Journal: Mediators of Inflammation

Article Title: Selenoprotein S Attenuates Tumor Necrosis Factor- α -Induced Dysfunction in Endothelial Cells

doi: 10.1155/2018/1625414

Figure Lengend Snippet: Effect of SelS on the viability of HUVECs and NO and eNOS expressions. (a) Viability of the transfected HUVECs after a 12 h stimulation with TNF- α (100 ng/ml), tested using the CCK-8 method. (b) The concentrations of NO in transfected HUVECs with 6 h TNF- α (10 ng/ml) treatment, determined using the nitrate reductase assay. The protein expression of eNOS in 10 ng/ml TNF- α -induced HUVECs after transfection with SelS plasmid (c) or SelS siRNA (d) examined by the western blot. The mRNA levels of eNOS in 10 ng/ml TNF- α -induced HUVECs after transfection with SelS plasmid (e) or SelS siRNA (f). The cells were transfected with pcDNA3.1-SelS recombinant plasmid or SelS siRNAs for 30 h. The eNOS expression was tested after 6 h TNF- α treatment. The results are representative of triplicate independent experiments and are presented as mean ± SD, ( n = 3). ∗∗ p < 0.01 versus control; # p < 0.05 versus empty vector; ## p < 0.01 versus empty vector; && p < 0.01 versus negative siRNA. CCK-8: cell counting kit-8; NO: nitric oxide; eNOS: endothelial nitric oxide synthase; E-vector: empty vector; Pc-SelS: pcDNA3.1-SelS plasmid; Neg.RNA: negative siRNA.

Article Snippet: Nitrocellulose membranes (Millipore, USA) containing the transferred proteins were soaked in blocking buffer for 2 h and then separately incubated at 4°C overnight with the following primary antibodies: SelS (Sigma, USA), endothelial nitric oxide synthase (eNOS; Proteintech, Wuhan, China), p-c-jun (Proteintech, Wuhan, China), c-jun (Proteintech, Wuhan, China), p-p38 MAPK (Abcam, USA), p38 MAPK (Abcam, USA), inhibitory kappa B α kinase β (IKK β , CST, USA), p-IKK β (CST, USA), inhibitory kappa B α (I κ B α , CST, USA), p-I κ B α (CST, USA), NF- κ B p65 (Bioworld, USA), Lamin B (Proteintech, Wuhan, China), and GAPDH (Proteintech, Wuhan, China).

Techniques: Transfection, CCK-8 Assay, Reductase Assay, Expressing, Plasmid Preparation, Western Blot, Recombinant, Control, Cell Counting

Journal: Journal of Functional Foods

Article Title: Fucoidan ameliorates diabetic skeletal muscle atrophy through PI3K/Akt pathway

doi: 10.1016/j.jff.2024.106076

Figure Lengend Snippet: Fig. 7. Fucoidan ameliorates glucose metabolism in diabetic skeletal muscle and PA treated C2C12 myotubes through activating PI3K/Akt. (A) Muscle glycogen content of gastrocnemius muscle. (B) PAS staining in the gastrocnemius muscle. Magnification, ×200. Scale bar, 50 μm. (C) Muscle glycogen area in the gastroc nemius muscle. (D) Relative mRNA level of GLUT4, GSK-3β and GS. (E–F) Western blot and quantification of GLUT4, GSK-3β and GS in gastrocnemius muscle. (G) Glycogen content in C2C12 cells. (H) The different concentrations of PA treated glucose consumption of C2C12 myotubes. (I) Glucose consumption in C2C12 cells. (J) Relative mRNA level of GLUT4, GSK-3β and GS in C2C12 cells. (K–L) Western blot and quantification of GLUT4, GSK-3β and GS in gastrocnemius muscle. Data are expressed as means ± SD (n = 3). *Р < 0.05, as compared to the NC group, #Р < 0.05, as compared to the DM or PA group. &Р < 0.05, as compared to the PA + Fu group.

Article Snippet: The membrane was incubated with primary antibody for 1.5 h, including PI3K p85 (dilution 1:4000; cat# 60225-1-Ig; Proteintech, Wuhan, China), Akt (dilution 1:5000; cat# 60203-2-Ig; Proteintech, Wuhan, China), p-Akt (dilution 1:5000; cat# 60225-1-Ig; Proteintech, Wuhan, China), mTOR (dilution 1:2000; cat# ab32028; Abcam, Cambridge, MA, United States), p-mTOR (dilution 1:1000; cat# ab109268; Abcam, Cambridge, MA, United States), p70S6K (dilution 1:1000; cat# ER31205; Huabio, Hangzhou, China), p-p70S6K (dilution 1:1000; cat# 34475S; Cell Signaling Technology, Danvers, MA, United States), GLUT4 (dilution 1:2000; cat# 66846-1-Ig; Proteintech, Wuhan, China), GSK-3β (dilution 1:2000; cat# ET1607-71; Huabio, Hangzhou, China), p-GSK-3β (dilution 1:2000; cat# ET1607-60; Huabio, Hangzhou, China), GS (dilution 1:1000; cat# ET1611-59; Huabio, Hangzhou, China), FoxO1 (dilution 1:3000; cat# 18592-1-AP; Proteintech, Wuhan, China), Atrogin-1 (dilution 1:1000; cat# ET7109-25; Huabio, Hangzhou, China), MuRF1 (dilution 1:2000; cat# HA500057; Huabio, Hangzhou, China) and β-actin (dilution 1:50000; cat# ET1701-80; Huabio, Hangzhou, China).

Techniques: Staining, Western Blot

Journal: Frontiers in cell and developmental biology

Article Title: Inhibiting Heat Shock Protein 90 Protects Nucleus Pulposus-Derived Stem/Progenitor Cells From Compression-Induced Necroptosis and Apoptosis.

doi: 10.3389/fcell.2020.00685

Figure Lengend Snippet: FIGURE 1 | Compression induced necrotic cell death of NPSCs. (A) IHC staining of Tie2 for marking NPSCs in non-degenerated (37 years old, male, grade II) and degenerated (43 years old, male, grade IV) human NP tissues (original magnification: ×400). (B) Cell viability of NPSCs examined by CCK-8 assays. (C) The relative release of LDH at different time points. (D) Representative dot plots of PI staining obtained from flow cytometry analysis of NPSCs. (E) The statistical analysis of PI positive ratio of NPSCs. (F) The morphological ultrastructural appearance of NPSCs observed by TEM. The NPSCs exposed to 48 h of compression displayed necrotic morphological changes, such as severe vacuolation, swelling of organelles and disruption of the plasma membrane. The data were expressed as mean ± SD from at least three independent experiments, and they were analyzed by a two-tailed t-test or ANOVA. (**P < 0.01, ***P < 0.001 vs. 0 h).

Article Snippet: Cells were then permeabilized with 0·5% Triton X-100 (Beyotime) for 15 min at room temperature (for staining of Tie2, the permeabilization was not performed), and blocked with goat serum albumin for 1 h. Next, samples were washed with Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 August 2020 | Volume 8 | Article 685 PBS and incubated with the mixture of mouse anti-HSP90 antibody (1:200, Santa Cruz Biotechnology) and rabbit antiphosphorylated MLKL (P-MLKL) antibody (Ser358, 1:200, Affinity Biosciences, OH, United States), the rabbit anti-HSP70 antibody (1:500, ABclonal, Wuhan, China), or rabbit anti-Tie2 antibody (1:100, Proteintech Group, Wuhan, China) at 4◦C overnight, followed by incubation with fluorophore-conjugated secondary antibody (1:200, Proteintech Group).

Techniques: Immunohistochemistry, CCK-8 Assay, Staining, Cytometry, Disruption, Clinical Proteomics, Membrane, Two Tailed Test

Journal: Frontiers in cell and developmental biology

Article Title: Inhibiting Heat Shock Protein 90 Protects Nucleus Pulposus-Derived Stem/Progenitor Cells From Compression-Induced Necroptosis and Apoptosis.

doi: 10.3389/fcell.2020.00685

Figure Lengend Snippet: FIGURE 9 | Inhibiting HSP90 attenuated the exhaustion of NPSCs in vivo. Hematoxylin and eosin staining (original magnification: ×25) and the IHC staining of Tie2 (original magnification: ×25 and 400) for labeling endogenous NPSCs of IVDs.

Article Snippet: Cells were then permeabilized with 0·5% Triton X-100 (Beyotime) for 15 min at room temperature (for staining of Tie2, the permeabilization was not performed), and blocked with goat serum albumin for 1 h. Next, samples were washed with Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 August 2020 | Volume 8 | Article 685 PBS and incubated with the mixture of mouse anti-HSP90 antibody (1:200, Santa Cruz Biotechnology) and rabbit antiphosphorylated MLKL (P-MLKL) antibody (Ser358, 1:200, Affinity Biosciences, OH, United States), the rabbit anti-HSP70 antibody (1:500, ABclonal, Wuhan, China), or rabbit anti-Tie2 antibody (1:100, Proteintech Group, Wuhan, China) at 4◦C overnight, followed by incubation with fluorophore-conjugated secondary antibody (1:200, Proteintech Group).

Techniques: In Vivo, Staining, Immunohistochemistry, Labeling