br Scratch wound healing assay
2.4. Scratch wound healing assay
MDA-MB-231 CP-456773 were grown in 35-mm Petri dishes until con-fluence. Then, a wound was created using a sterile 200 μl pipette tip. Cells were treated or not with chloroquine and/or astemizole and mi-gration analysis was performed after 16 and 20 h incubation at 37 °C. Photographs were taken immediately or at diﬀerent times of wound closure using an inverted microscope (Nikon, Japan) and measure with the Image J software (Schneider et al., 2012). Cell migration was cal-culated by quantifying the percentage change in area: (original scratch area – new scratch area)/original scratch area x 100.
2.5. Data analysis and statistics
Patch-clamp data were processed using Clampfit 10 (Molecular Devices) and analyzed in Origin 8.6 (OriginLab Corp. Northampton, MA, USA).
The statistical analysis was carried out by using SPSS statistics 22.0 software (IBM Corporation, Armonk, NY, USA). Data are presented as the mean ± SEM. For electrophysiological experiments, statistical comparisons were made by unpaired or one-sample Student's t-test. For cell migration, the diﬀerence between groups were compared by one-way ANOVA followed by post hoc Dunnett's test. Statistical significance was set at p < 0.05.
The eﬀects of chloroquine were first examined using the whole-cell configuration of the patch clamp technique. Kv10.1 currents were eli-cited by 500-ms depolarizing steps to +60 mV, followed by repolar-ization to −70 mV. The voltage protocol was repeated every 10-s. Kv10.1 channels were inhibited by chloroquine in a concentration-de-pendent manner (Fig. 1). Fig. 1A shows a temporal course of Kv10.1 currents in response to external application of 30 μM chloroquine.
Fig. 1. Eﬀect of chloroquine (CQ) on Kv10.1 channels expressed in HEK293 cells and re-corded in whole-cell configuration. (A) Representative time course of the eﬀect of 30 μM chloroquine on Kv10.1 currents. (B) Representative Kv10.1 current traces in absence (control) and presence of chloroquine at in-creasing concentrations (3–300 μM). (C) Concentration-response relationship for Kv10.1 current inhibition by chloroquine at +60 mV. Mean values were plotted against chloroquine concentration and fitted with the Hill equation. Mean IC50 was 31.05 ± 4.5 μM and the Hill coeﬃcient, nH = 0.88 ± 0.1 (n = 7).
Fig. 2. Block of Kv10.1 currents by chloroquine (CQ) is voltage-dependent. (A) Representative Kv10.1current traces obtained with the protocol shown below panel B. (B) Currents recorded in presence of 30 μM chloroquine in the same cell depicted in panel A. inset, superimposed tail currents obtained at −50 mV after a 500-ms depolarization to +80 mV. (C) Normalized current-voltage re-lationship for currents measures at the end of the 500-ms pulses in control and in the presence of chloroquine (n = 9). (D) Fractional block (1 – Idrug/Icontrol) of Kv10.1 currents plotted as a function of test potential (n = 9). Idrug and Icontrol were measured at the end of the depolarizing pulse at each membrane poten-tial.
We further explored the eﬀect of chloroquine on Kv10.1 by re-cording currents in a voltage range from −60 to +80 mV, applied in 10-mV increments from a holding potential of −80 mV. In the absence of drug, Kv10.1 currents slowly activate and does not inactivate (Fig. 2A). In the presence of drug (30 μM chloroquine), the currents are profoundly inhibited and showed a crossover at potentials above +50 mV (Fig. 2B). Plots of the maximum current amplitudes measured at the end of the 500-ms pulses indicate Pleiotropic gene block by chloroquine (30 μM) was voltage-dependent with more pronounced reductions at more depolarized potentials (Fig. 2C–D).
The inset in Fig. 2A shows the superposition of the tail currents obtained at −50 mV after a 500-ms depolarization to +80 mV. The tail current declined slower in the presence of chloroquine compared to that of control, resulting in the crossover phenomenon.