Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: HUVECs were incubated with AngII at different concentrations for different times. Ang II time (A)- or concentration (B)-dependently enhanced the Kv1.5 protein expression. Pretreatment with MT for 30 min inhibited Ang II (2 µM, 24 h)-induced increase in intracellular ROS levels in HUVECs in a concentration-dependent manner (C), as determined by DCF fluorescence. MT also significantly attenuated Ang II (2 µM, 24 h)-induced HUVEC injury. Incubation of HPAECs with oxLDL at the concentration of 37.5, 75 and 150 µg/ml significantly increased EC injury (D), intracellular ROS production (E) and Kv1.5 protein expression (F) in a concentration-dependent manner. The values were presented as ± SEM of 6 independent experiments for Ang II- or oxLDL-treatment, respectively. * P <0.05, ** P <0.01 vs. control; $ P <0.05, # P <0.01 vs. Ang II group.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Incubation, Concentration Assay, Expressing, Fluorescence, Control

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: ( A ). HPAECs were treated with 2.5, 5, 10 nM Kv1.5 siRNA for 48 h and Kv1.5 protein expression was analyzed by western blot. * P <0.05, ** P <0.01 vs. Vehicle control, n = 3. ( B ). HPAECs were infected with adnovirus containing KCNA5 (Ad-Kv1.5) and control–adnovirus (Ad-con) for 12, 24, 48 h. * P <0.05, ** P <0.01 vs. Ad-con at corresponding time points, n = 3.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Expressing, Western Blot, Control, Infection

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: HPAECs were transfected with Kv1.5 siRNA (10 nM) or control siRNA for 48 h, or infected with adnovirus containing KCNA5 and control–adnovirus for 24 h, then treated with oxLDL (150 µg/ml) for further 24 h. The oxLDL-induced EC injury was detected by optical microscopy and DAPI staining. The optical microscope observation (A) and DAPI staining (B) show the morphological changes in Kv1.5 siRNA pretreated HPAECs. The mean values of percentages of apoptotic cells were summarized after DAPI staining in Kv1.5 siRNA (C)- and KCNA5 adnovirus- pretreated (D) HPAECs. The values are presented as means ± SEM of 6 independent experiments. * P <0.05, ** P <0.01 vs. Vehicle control; # P <0.05, ## P <0.01 vs. Ad-con.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Transfection, Control, Infection, Microscopy, Staining

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: Intracellular ROS levels were detected using DCFH-DA staining in HPAECs transfected with Kv1.5 siRNA (10 nM) or infected with adnovirus containing KCNA5 , which were treated with oxLDL (150 µg/ml) for further 1 h. ( A ) & ( B ). Kv1.5 siRNA significantly attenuated oxLDL-induced endothelial ROS overproduction in HPAECs, as demonstrated by representative images from laser scanning confocal microscopy and bar graph showing fluorescence intensity measured in Multi-Mode Microplate Reader. ( C ). Bar graph shows that adnoviral KCNA5 gene transfer exaggerated oxLDL-induced endothelial ROS overproduction in HPAECs. The values are presented as means ± SEM of 6 independent experiments. * P <0.05, ** P <0.01 vs. Vehicle control; # P <0.05, ## P <0.01 vs. Ad-con.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Staining, Transfection, Infection, Confocal Microscopy, Fluorescence, Control

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: The mitochondrial ROS generation was detected using MitoSOX reagents in HPAECs transfected with Kv1.5 siRNA (10 nM) or infected with adnovirus containing KCNA5 , which were treated with oxLDL (150 µg/ml) for further 1 h. ( A ) The representative images from laser scanning confocal microscopy Kv1.5 siRNA significantly attenuated oxLDL-induced mitochondrial superoxide production in HPAECs. ( B ) & ( C ). The mitochondrial ROS levels in HPAECs transfected with Kv1.5 siRNA (B) or infected with adnovirus overexpression of Kv1.5 (C) were quantified by the fluorescent plate reader and expressed as the ratio of MitoSOX to Mitotracker. The values are presented as means ± SEM of 6 independent experiments. * P <0.05, ** P <0.01 vs. Vehicle control; # P <0.05, ## P <0.01 vs. Ad-con.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Transfection, Infection, Confocal Microscopy, Over Expression, Control

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: The UCP2 protein expression was detected by western blot in HPAECs transfected with Kv1.5-siRNA (A) and or infected with adnovirus containing KCNA5 (B). Digital photographs showing UCP2 protein expression are on the top panel and bar graph showing quantitative analysis of the total protein are on the lower panel. The values are presented as means ± SEM of 6 independent experiments. ** P <0.01 vs. corresponding control group; # P <0.01 vs. oxLDL group.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Expressing, Western Blot, Transfection, Infection, Control

Journal: PLOS ONE

Article Title: Hydrogen decreases susceptibility to AngII-induced atrial fibrillation and atrial fibrosis via the NOX4/ROS/NLRP3 and TGF-β1/Smad2/3 signaling pathways

doi: 10.1371/journal.pone.0310852

Figure Lengend Snippet: (A) Western blot bands showing p-CaMKII together with p-RyR2. (B) and (C) The relative p-CaMKII and p-RyR2 protein expression. (D) Western blot bands of Kv1.5, Kir3.1 and Kir3.4. (E-G) The relative levels of Kv1.5, Kir3.1 and Kir3.4 proteins; (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus Con group; #P < 0.05, ##P < 0.01, ###P < 0.001, versus AngII group.

Article Snippet: Thereafter, primary antibodies, which included NOX4 (14347-1-AP, Proteintech, 1:1000), p22phox (sc-271968, Santa Cruz Biotechnology, 1:200), NLRP3 (ab214185, Abcam, 1:1000), ASC (sc-514414, Santa Cruz Biotechnology, 1:200), caspase1-p20 (22915–1-AP, Santa Cruz Biotechnology, 1:1,000), phospho-CaMKII (ab182674, Abcam, 1:1000), phospho-RyR2 (Ser2814,GTX00626, Genetex, 1:1000), Kv1.5 (YT2507, ImmunoWay, 1:1000), Kir3.1 (YT2475, ImmunoWay, 1:1000), Kir3.4 (YT2475, ImmunoWay, 1:1000), TGF-β1 (ab215715, Abcam, 1:1000), phospho-TGFβR I (YP1191, ImmunoWay; 1:1000), phospho-TGFβR II (A1415, Abclonal, 1:1000), phospho-Smad2 (YP1185, ImmunoWay; 1:1000), phospho-Smad3 (YP0585, ImmunoWay; 1:1000), α-SMA (bs-10196R, Bioss, 1:1000), collagen I (bs-0578R, Bioss, 1:500), collagen III (bs-0549R,Bioss, 1:500), and GAPDH (10494-1-AP, Proteintech, 1:10000), were added for overnight membrane incubation under 4°C in the shaker.

Techniques: Western Blot, Expressing

Journal: PLOS ONE

Article Title: Hydrogen decreases susceptibility to AngII-induced atrial fibrillation and atrial fibrosis via the NOX4/ROS/NLRP3 and TGF-β1/Smad2/3 signaling pathways

doi: 10.1371/journal.pone.0310852

Figure Lengend Snippet: (A) ROS production in HL-1 cells by immunofluorescence staining (×200). (B) Expression of NLRP3 within HL-1 cells by immunofluorescence staining (×200). (C) Intracellular Ca 2+ concentration in HL cells by fluorescent probes (×200). (D), (E) and (F) mRNAs of Kv1.5, Kir3.1 and Kir3.4 by qRT-PCR. (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, versus Con group; # P < 0.05, ## P < 0.01, ### P < 0.001, versus AngII group.

Article Snippet: Thereafter, primary antibodies, which included NOX4 (14347-1-AP, Proteintech, 1:1000), p22phox (sc-271968, Santa Cruz Biotechnology, 1:200), NLRP3 (ab214185, Abcam, 1:1000), ASC (sc-514414, Santa Cruz Biotechnology, 1:200), caspase1-p20 (22915–1-AP, Santa Cruz Biotechnology, 1:1,000), phospho-CaMKII (ab182674, Abcam, 1:1000), phospho-RyR2 (Ser2814,GTX00626, Genetex, 1:1000), Kv1.5 (YT2507, ImmunoWay, 1:1000), Kir3.1 (YT2475, ImmunoWay, 1:1000), Kir3.4 (YT2475, ImmunoWay, 1:1000), TGF-β1 (ab215715, Abcam, 1:1000), phospho-TGFβR I (YP1191, ImmunoWay; 1:1000), phospho-TGFβR II (A1415, Abclonal, 1:1000), phospho-Smad2 (YP1185, ImmunoWay; 1:1000), phospho-Smad3 (YP0585, ImmunoWay; 1:1000), α-SMA (bs-10196R, Bioss, 1:1000), collagen I (bs-0578R, Bioss, 1:500), collagen III (bs-0549R,Bioss, 1:500), and GAPDH (10494-1-AP, Proteintech, 1:10000), were added for overnight membrane incubation under 4°C in the shaker.

Techniques: Immunofluorescence, Staining, Expressing, Concentration Assay, Quantitative RT-PCR