Journal: Physiological Reports

Article Title: Extracellular acidity and ATP modulate ion currents in human cumulus cells indicating possible roles as metabolic sensors of the follicular microenvironment

doi: 10.14814/phy2.70729

Figure Lengend Snippet: CC‐type 1 expresses Kv1.5 potassium currents. (a) I–V relationship between the peak current and at 100 ms ( n = 23, seven independent experiments). Inset: Representative current traces family used to build the I–V relationship by using the protocol described in Figure . The red dashed line represents I = 0 pA. (b) The voltage‐dependent inactivation was studied by plotting the peak of the test current as a function of the pre‐conditioning potential, interpolating the values obtained according to the Boltzmann equation (Equation ), with V / 2 = −55.7 mV and k = 13.9 ( n = 23, seven independent experiments). Inset: Representative steady state current inactivation obtained by conditioning the cell from −110 to −40 mV with an incremental test pulse of 20 mV. (c) Sensitivity of potassium currents before (CTRL, black trace) and after (red traces) application of 10 μM PAP‐1 (three independent experiments). (d) Sensitivity of potassium currents before (CTRL, black trace) and after (red traces) application of 200 μM TMA (three independent experiments). (e) Sensitivity of potassium currents before (CTRL, black trace) and after (red traces) application of 1 mM 4‐AP (three independent experiments). Inset (c–e): Protocol used to study pharmacology profile by pre‐conditioning the cell at −80 mV for 500 ms, then bringing it to +140 mV from a V holding potential (−60 mV). (f) Fluorescence imaging by immunocytochemistry revealed the expression and localization of K V 1.5 channels in cumulus cells (three independent experiments). K V 1.5 channel expression was obtained by using primary rabbit IgG polyclonal antibody FITC‐conjugated (1:100). (g) Nuclei were stained with DAPI (1:1000). (h) Merged with Ab anti‐KCNA5‐FITC conjugated and DAPI is displayed. Scale bar represents 20 μm.

Article Snippet: Cells were then washed and incubated at 4°C overnight with primary rabbit polyclonal antibodies anti‐KCNMA1 (ThermoFisher, cat. no. PA1‐923), anti‐TRPM5 (Alomone Labs, cat. no. ACC‐045) or anti‐KCNA5 FITC‐conjugated (Alomone Labs, cat. no. APC‐150‐F), diluted 1:100 in PBS.

Techniques: Fluorescence, Imaging, Immunocytochemistry, Expressing, Staining

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