Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: The Pro-Tumoral Activity of Heparan Sulfate 3- O -Sulfotransferase 3B (HS3ST3B) in Breast Cancer MDA-MB-231 Cells Is Dependent on the Expression of Neuropilin-1

doi: 10.3390/molecules23102718

Figure Lengend Snippet: Stable expression of HS3ST3B in MDA-MB-231 cells. Cells were transfected with the expression vector encoding HS3ST3B and then cultured in complete DMEM medium in the presence of 400 µg/mL G418. After 14 days of culture, individual colonies were isolated by limit dilution and amplified in medium supplemented with G418. In parallel, cells were transfected with the empty vector to obtain the control parental cells (pEmpty). ( A ) Following RNA extraction, the mRNA level of HS3ST3B was quantified by real-time RT-PCR in each clone. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD of triplicates. ( B ) HS3ST3B expression in the clones C and D was analyzed by confocal microscopy. To this end, cells were seeded on glass coverslips, permeabilized and then incubated in the presence of anti-HS3ST3B antibodies. After wash, they were immunostained with secondary antibodies conjugated to Alexa-568, in order to highlight the enzyme in red fluorescence. For the detection of 3- O -sulfated motifs, recombinant HSV-1 gD (10 μg/mL) was incubated with primary anti-gD antibody for 30 min at 4 °C, and the immune complex was added to cells for an additional 30 min-incubation. After washing, cells were fixed and incubated for 1 h with Alexa 488-conjugated secondary antibody (green fluorescence). In all the microscopy experiments, nuclei were stained in blue with DAPI, in order to visualize cell nuclei ( N = 3 separate experiments; n = 30 cells). Scale bar = 10 µm.

Article Snippet: Transfection was performed with Lipofectamine ® 2000, according to the manufacturer’s instructions (Thermo Fisher), after which cells were cultured in culture medium in the presence of 400 μg/mL G418 (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Isolation, Amplification, Control, RNA Extraction, Quantitative RT-PCR, Clone Assay, Confocal Microscopy, Incubation, Fluorescence, Recombinant, Microscopy, Staining

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Effect of Klotho on cellular viability in HUVECs. Cells were exposed to various concentrations of ox-LDL (25, 50, 100, and 200 μg/ml) for 24 h. a HUVEC morphology was observed under an inverted phase contrast microscope (×10) following 24 h of ox-LDL treatment. Typical cellular fragmentations, vacuoles, and debris are arrowed. (Bar = 50 μm) ( b ) Cellular viability was detected by MTT assay. c and d SOD enzymatic activity and MDA levels in HUVECs were analyzed using commercially available assay kits. e HUVECs were pretreated with 100, 200, 400, and 800 pM of Klotho for 1 h, and then incubated with ox-LDL (50 μg/ml) for 24 h. Cellular viability was detected by MTT assay. Data are shown as mean ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Microscopy, MTT Assay, Activity Assay, Incubation, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho inhibited ROS production induced by ox-LDL in HUVECs. HUVECs were pre-incubated with 200 pM of recombinant human Klotho for 1 h, then treated with ox-LDL (50 μg/mL) for another 24 h. Ox-LDL alone and Klotho alone were used as controls. a Images observed under an inverted fluorescence microscope. (Bar = 50 μm) ( b ) Output figure of the fluorescence intensity detected by flow cytometry. c Average fluorescence intensity: Mean = total area under the peak/the total number of cells. Data are shown as mean ± S.D. d Lipid peroxidation was assessed by measuring the MDA levels in HUVECs treated with ox-LDL and/or Klotho ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. blank control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Incubation, Recombinant, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: SOD, Cu/Zn-SODandgp91 phox in ox-LDL and Klotho-treated HUVECs. Cellular treatment was the same as described in Fig. . a Intracellular SOD activity was determined by the hydroxylamine method. b , c and d Western blotanalysis of Cu/Zn-SOD and gp91 phoxexpression in pre-treated HUVECs. Densitometry of the probed bands from the western blot were analyzed by Image J2x. Values are means ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Activity Assay, Western Blot, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho regulated NO production in HUVECs. a NO production in pre-treated HUVECs. b , c , d , and e mRNA levels of iNOS, eNOS, PI3K, and Akt were measured by real time PCR. Values are means ± S.D. ( n = 3). Statistical differences are expressed as # p < 0.05 vs . control; * p < 0.05 vs . ox-LDL. ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: eFood

Article Title: Docosahexaenoic Acid and Nervonic Acid Synergically Enhance Cognitive Memory in Normal Mice via Brain Fatty Acids Remodeling

doi: 10.1002/efd2.70059

Figure Lengend Snippet: FIGURE 3 | DHA and NA enhanced c‐Fos expression in the hippocampus of normal mice. (A) Representative c‐Fos immunopanned photos in the hippocampus. (B) The number of c‐Fos+ neurons in the hippocampal DG area. (C) The expression of PSD95 in the hippocampus. (D) The expression of synaptophysin (SYP) in the hippocampus. *p < 0.05, **p < 0.01 vs control group. One‐way ANOVA post‐hoc Dunnett's test (mean ± SEM, n = 4).

Article Snippet: Immunostaining was performed using monoclonal antibodies against c‐Fos (1:1000; Cell Signaling Technology, cat#2250S) and synaptophysin (SYP, 1:400; Santa Cruz, sc‐17750), polyclonal antibody against PSD95 (1:200; Invitrogen, cat#51‐6900), followed by incubation at 4°C for 24 h. Goat anti‐ mouse (Alexa Fluor 594, 1:500, ab150116) and goat anti‐rabbit (Alexa Fluor 488, 1:500, ab150077) IgG were used as secondary antibodies and incubated in the dark at room temperature for 2 h. Fluorescent images were captured using an ECHO Revlove fluorescence microscope (ECHO, San Diego, California, USA) and quantitatively analyzed using ImageJ software (NIH, Bethesda, Maryland, USA).

Techniques: Expressing, Control