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:

Article Title: Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior to de Novo Vesicle Formation *

doi: 10.1074/jbc.M109134200

Figure Lengend Snippet: Wild type (SEY6210) (A and B) or aut7Δ (WPHYD7) (C) cells were co-transformed with the following pairs of copper-inducible plasmids. A, YFP-Aut7 (pCuYFPAUT7(426)) and CFP-Cvt9 (pCuCFPCVT9(424)); B, YFP-Apg12 (pCuYFPAPG12(426)) and CFP-Cvt9; C, Apg5-YFP (pAPG5YFP(426)) and CFP-Cvt9. The transformed cells were grown to midlog stage in SMD, induced with 30 μm CuSO4 for 2 h (A and B) or with 5 μm CuSO4 for 30 min (C), and examined with a Nikon E-800 fluorescence microscope equipped with a Hamamatsu Orca 2 digital camera and processed with Openlab software. The YFP fusions to the vesicle component Aut7 and the conjugation components Apg12 and Apg5 co-localize with CFP-Cvt9. The DIC panels are images obtained with differential interference contrast optics.

Article Snippet: The transformed cells were grown to midlog stage in SMD, induced with 10 μ m CuSO 4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment.

Techniques: Transformation Assay, Fluorescence, Microscopy, Software, Conjugation Assay

Journal:

Article Title: Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior to de Novo Vesicle Formation *

doi: 10.1074/jbc.M109134200

Figure Lengend Snippet: Wild type cells (SEY6210) were co-transformed with the following pairs of plasmids. A, copper-inducible YFP-Cvt9 (pCuYFP-CVT9(426)) and Cvt19-CFP under endogenous promoter control (pCVT19CFP(414)); B, copper-inducible YFP-Aut7 (pCuYFPAUT7(426)) and Cvt19-CFP under endogenous promoter control (pCVT19CFP(414)). The transformed cells were grown to midlog stage in SMD, induced with 10 μm CuSO4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in Fig. 1 and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment. DIC, differential interference contrast.

Article Snippet: The transformed cells were grown to midlog stage in SMD, induced with 10 μ m CuSO 4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment.

Techniques: Transformation Assay, Fluorescence, Microscopy

Journal:

Article Title: Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior to de Novo Vesicle Formation *

doi: 10.1074/jbc.M109134200

Figure Lengend Snippet: Wild type cells (SEY6210) were co-transformed with plasmids, under CUP1 copper-inducible control, expressing either YFP-Apg9 (pCuYFPAPG9(426)) and CFP-Cvt9 (pCuCFPCVT9(424)) (A), YFP-Apg9 and Cvt19-CFP (pCVT19CFP(414)) (B), or YFP-Aut7 (pCuYFPAUT7(426)) and CFP-Apg9 (pCuCFPAPG9(424)) (C). Transformed cells were grown to midlog stage in SMD and induced for 1–2 h with 30 μm CuSO4. Images were taken and examined with a Nikon E-800 fluorescence microscope as described in Fig. 1 and under “Experimental Procedures.” Apg9 displays multiple punctate dots when co-expressed with Cvt19 and Aut7 but only a single dot when co-expressed with Cvt9. The YFP-Apg9 and CFP-Cvt9 dots co-localize. DIC, differential interference contrast.

Article Snippet: The transformed cells were grown to midlog stage in SMD, induced with 10 μ m CuSO 4 for 2 h, and examined with a Nikon E-800 fluorescence microscope as described in and under “Experimental Procedures.” A fluorescent protein fusion of Cvt19 co-localizes with both Cvt9 and Aut7 at the perivacuolar compartment.

Techniques: Transformation Assay, Expressing, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Reversible photoregulation of cell-cell adhesions with opto-E-cadherin

doi: 10.1038/s41467-023-41932-0

Figure Lengend Snippet: a Schematic representation of opto-E-cad-dependent cell signalling. In the dark, the opto-E-cads on adjacent cells homophilically bind to each other, and the catenins (p120, α- and β) are recruited to the intracellular tail and link it to F-actin. Under blue light, opto-E-cads do not form cell–cell adhesions, no catenins are recruited and there is no link to the actin cytoskeleton. b Confocal fluorescence microscopy images of opto-E-cad-MDA cells in the dark or under blue light (MDA-MB-231 and MCF-7 cells in 2D cultures). F-actin is shown in red, nuclei are shown in blue and p120 is shown in yellow. Scale bars are 50 µm and 15 µm for the inset. c Cell spreading area of opto-E-cad cells forming cell–cell adhesions in cluster compared to single cells. n = 35, 46, 14, 46, 46. Boxplots show median, 25th, and 75th percentile. The lower and upper boundaries of whiskers indicate the minima and maxima, respectively. All comparisons are performed using Fisher’s One Way ANOVA test and p < 0.05 was treated as the significance threshold. d 3D reconstruction and maximal intensity projection for p120 (yellow) and nuclei (blue) of opto-E-cad-MDA cell clusters after 120 min in the dark. Scale bar is 50 µm ( n = 3). e Western blot of E-cadherin, p120 and β-actin (loading control) for opto-E-cad-MDA in the dark or under blue light and E-cad-MDA and MDA-MB-231 cells as positive and negative control, respectively ( n = 4). f RT-PCR results for opto-E-cad-MDA in the dark and under blue light for EMT markers. (two-tailed t-test, n = 12 and 10). Data are represented as individual values ± SD. Source data are provided as a Source Data file.

Article Snippet: Subsequently, the cells were washed three times with PBS, blocked with 1% BSA (Sigma-Aldrich # A7030-100G) in PBS for 1 h, and incubated with the primary rabbit-anti-delta-catenin (p120 catenin, cell signalling #59854, diluted 1:800) or the primary mouse-anti-E-cadherin antibody (cell signalling #14472, diluted 1:200) in 1% BSA in PBS overnight at 4 °C.

Techniques: Fluorescence, Microscopy, Western Blot, Control, Negative Control, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test