Coexpression of B-RAF increased hERG current in <i>Xenopus</i> oocytes.

<p><b>A.</b> Original tracings recorded in <i>Xenopus</i> oocytes injected with water (a), with cRNA encoding hERG alone (b) or with cRNA encoding hERG together with wild-type B-RAF (c). The <i>Xenopus</i> oocytes were depolarized from −80 mV holding potential to different voltages followed by a 500 ms repolarization to −60 mV evoking outward tail currents. <b>B.</b> Arithmetic means ± SEM (n = 12–47, arbitrary units) of the normalized outward tail current following a depolarization to +70 mV, recorded in <i>Xenopus</i> oocytes injected with water (white bar), with cRNA encoding hERG alone (light grey bar), or with cRNA encoding both, hERG and wild-type B-RAF (black bar). ***(p<0.001) indicates statistically significant difference from <i>Xenopus</i> oocytes expressing hERG channels alone. <b>C.</b> Arithmetic means ± SEM (n = 12–47, nA) of the peak tail current as a function of voltage in <i>Xenopus</i> oocytes injected with water (black triangles), with cRNA encoding hERG alone (white circles) or with cRNA encoding hERG and wild-type B-RAF (black circles). <b>D.</b> Arithmetic means ± SEM (n = 22–47, arbitrary units) of the normalized peak tail current as a function of voltage in <i>Xenopus</i> oocytes injected with cRNA encoding hERG alone (white circles) or with cRNA encoding hERG together with wild-type B-RAF (black circles).</p

Coexpression of B-RAF increased hERG-HA protein abundance at the surface of hERG-expressing <i>Xenopus</i> oocytes.

<p><b>A.</b> Confocal images of hERG-HA protein cell surface expression in <i>Xenopus</i> oocytes injected with water (left panel), with cRNA encoding hERG-HA alone (middle panel) or with cRNA encoding hERG-HA together with wild-type B-RAF (right panel). Images are representative of three independent experiments. <b>B.</b> Arithmetic means ± SEM (n = 81–93, arbitrary units) of hERG-HA protein abundance in the cell membrane measured by chemiluminescence in <i>Xenopus</i> oocytes injected with water (white bar), with cRNA encoding hERG-HA alone (light grey bar), or cRNA encoding hERG-HA and wild-type B-RAF (black bar). ***(p<0.001) indicates statistically significant difference from <i>Xenopus</i> oocytes expressing hERG channels alone.</p

B-RAF inhibitor PLX4720 decreased hERG currents in rhabdomyosarcoma RD cells.

<p><b>A.</b> Inward currents elicited in a bath solution containing 40: the membrane potential was held at −80 mV and then after the preconditioning step from −80 mV to +60 mV for 2 s stepped to the test potential of −120 mV for 500 ms. The currents were measured in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (a) or with 10 µM B-RAF inhibitor PLX-4720 (b). <b>B.</b> Mean peak current density ± SEM (n = 5–12) plotted against the precondition potential in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white cycles) or with 10 µM B-RAF inhibitor PLX-4720 (black cycles). C. Mean peak current density ± SEM (n = 5–12) measured at −120 mV after the precondition potential to +50 mV in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone.</p

B-RAF inhibitor PLX-4720 decreased hERG protein abundance at the cell surface in rhabdomyosarcoma RD cells.

<p><b>A.</b> Representative original western blot showing hERG membrane protein abundance (anti-K_v11.1 antibody, Alamone Labs) analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720). <b>B.</b> Arithmetic means ± SEM (n = 7, arbitrary units) of normalized hERG membrane protein abundance analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone. <b>C.</b> Representative original dot plots of hERG-FITC positive cells at the cell surface analysed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720); FL-1 Height: hERG-FITC fluorescence intensity. <b>D.</b> Arithmetic means ± SEM (n = 5, %) of normalized percentage of positive cells showing hERG expression at the cell surface analyzed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone.</p

ß-catenin increases HERG current.

<p><b>A.</b> Original tracings recorded in oocytes injected with H₂0 (1), with cRNA encoding HERG (2) or HERG coexpressing ß-catenin (3). The oocytes were depolarized from −80 mV holding potential to different voltages followed by a 500 ms pulse to −60 mV evoking outward tail currents. The small insert displays the applied voltage protocol. <b>B.</b> Arithmetic means±SEM (n = 8–11) of the normalized outward tail current following a depolarization to +80 mV recorded in oocytes injected with H₂0 (left bar), with cRNA encoding HERG (middle bar) or with RNA encoding HERG and ß-catenin (right bar). * indicates statistical significance (p<0.05) from the absence of ß-catenin cRNA. <b>C.</b> IV curves of outward tail currents illustrated in B (upper panel) and IV curves of outward tail currents following normalization to the maximal tail current of the respective group (lower panel).</p

ß-catenin increases the surface abundance of HERG.

<p>Arithmetic means±SEM (n = 19–56) of the normalized HA-dependent surface chemiluminescence of oocytes injected with H₂0 (1^st bar), with cRNA encoding ß-catenin (2^nd bar), encoding HERG (3^rd bar) or encoding both, HERG and ß-catenin (4^th bar). ***indicates statistical significance (p<0.001) from the absence of ß-catenin cRNA.</p

The effect of Brefeldin A on ß-catenin-stimulated HERG currents.

<p>Arithmetic means±SEM (n = 15–19) of the normalized outward tail current following depolarization to +80 mV recorded in oocytes injected with cRNA encoding HERG (white bars) or with cRNA encoding HERG and ß-catenin (black bars), prior to (0 days) and following incubation for 24 hours (1 day) or 48 hours (2 days) with 5 µM Brefeldin A prior to the measurement. **, ***indicate statistical significance (p<0.01, p<0.001) from the absence of ß-catenin cRNA.</p

HERG currents are insensitive to truncated ß-catenin^1–530 and stimulated by N-cadherin.

<p>Arithmetic means ± SEM (n = 10–20) of the normalized outward tail current following depolarization to +80 mV and recorded in oocytes injected with water (white bar), or with cRNA encoding HERG (dark grey bar) with ß-catenin (first black bar) or with the truncated mutant ß-catenin^1–530 (second black bar), without and with co-expression of N-cadherin (dotted dark grey bars). *, ***indicate statistical significance (p<0.05, p<0.001) from the absence of HERG cRNA, # (p<0.05) indicates statistical significance from the absence of N-cadherin.</p

The effect of ß-catenin on HERG currents is not modified by actinomycin D.

<p>Arithmetic means±SEM (n = 9–13) of the normalized outward tail current following a depolarization to +80 mV recorded in oocytes injected with cRNA encoding HERG (white bars) or with RNA encoding HERG and ß-catenin (black bars), incubated for 24 hours without (left bars) or with (right bars) 10 µM actinomycin D prior to the measurement. *indicates statistical significance (p<0.05) from the absence of ß-catenin cRNA.</p

Impact of AMP-Activated Protein Kinase α1 Deficiency on Tissue Injury following Unilateral Ureteral Obstruction

<div><p>Background</p><p>AMP-activated protein kinase (Ampk) is a sensor of the cellular energy status and a powerful regulator of metabolism. Activation of Ampk was previously shown to participate in monocyte-to-fibroblast transition and matrix protein production in renal tissue. Thus, the present study explored whether the catalytic Ampkα1 isoform participates in the regulation of the renal fibrotic response following unilateral ureteral obstruction (UUO).</p><p>Methods</p><p>UUO was induced in gene-targeted mice lacking functional Ampkα1 (Ampkα1^-/-) and in corresponding wild-type mice (Ampkα1^+/+). In the obstructed kidney and, for comparison, in the non-obstructed control kidney, quantitative RT-PCR, Western blotting and immunostaining were employed to determine transcript levels and protein abundance, respectively.</p><p>Results</p><p>In Ampkα1^+/+ mice, UUO significantly up-regulated the protein abundance of the Ampkα1 isoform, but significantly down-regulated the Ampkα2 isoform in renal tissue. Phosphorylated Ampkα protein levels were significantly increased in obstructed kidney tissue of Ampkα1^+/+ mice but not of Ampkα1^-/- mice. Renal expression of α-smooth muscle actin was increased following UUO, an effect again less pronounced in Ampkα1^-/- mice than in Ampkα1^+/+ mice. Histological analysis did not reveal a profound effect of Ampkα1 deficiency on collagen 1 protein deposition. UUO significantly increased phosphorylated and total Tgf-ß-activated kinase 1 (Tak1) protein, as well as transcript levels of Tak1-downstream targets <i>c-Fos</i>, <i>Il6</i>, <i>Pai1</i> and <i>Snai1</i> in Ampkα1^+/+ mice, effects again significantly ameliorated in Ampkα1^-/- mice. Moreover, Ampkα1 deficiency inhibited the UUO-induced mRNA expression of <i>Cd206</i>, a marker of M2 macrophages and of <i>Cxcl16</i>, a pro-fibrotic chemokine associated with myeloid fibroblast formation. The effects of Ampkα1 deficiency during UUO were, however, paralleled by increased tubular injury and apoptosis.</p><p>Conclusions</p><p>Renal obstruction induces an isoform shift from Ampkα2 towards Ampkα1, which contributes to the signaling involved in cell survival and fibrosis.</p></div