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Figure Lengend Snippet: ROCK inhibitors enhance 2D colon cancer cell invasiveness dependently on Na V 1.5 channels. ( A ) Summary of SW620 colon cancer cell invasiveness studies performed either on Matrigel-coated (300 µg/mL) or Collagen I-coated (500 µg/mL) invasion inserts in control condition (vehicle, 0.1% DMSO), in presence of TTX (30 µM), Y-27632 (10 µM) or the combination Y-27632 + TTX. Results, from 7–8 independent experiments, are expressed relative to the CTL condition which appears as a dashed line. *** P < 0.001 compared to CTL condition (Mann–Whitney rank sum test); ## P < 0.01 compared to the condition performed in presence of TTX (Mann–Whitney rank sum test). ( B ) Summary of SW620 colon cancer cell invasiveness studies performed on Matrigel-coated (300 µg/mL) invasion inserts in control condition, in presence of TTX (30 µM), Fasudil (20 µM) or the combination Fasudil + TTX. Results, from 8 independent experiments, are expressed relative to the CTL condition (vehicle, 0.1% DMSO) which appears as a dashed line. *** P < 0.001 compared to CTL condition (Mann–Whitney rank sum test); ## P < 0.01 compared to the condition in presence of TTX (Mann–Whitney rank sum test). ( C ) Effect of silencing the expression of ROCK-1, or ROCK-2 or RhoA using specific siRNA (siROCK1, siROCK2 or siRhoA, respectively), compared to the transfection of irrelevant siRNA (siCTL), on SW620 colon cancer cell invasiveness. These experiments were performed in absence (CTL) or presence of TTX (30 µM). Results are expressed as ratios of mean results obtained with siCTL cells in CTL condition (vehicle). The dashed line indicates a ratio of 1. Results are from 12 to 15 independent experiments and were analysed using Mann–Whitney rank sum tests. *** P < 0.001 compared to CTL condition in siCTL cells; * P < 0.05 compared to CTL condition in siCTL cells; +++ P < 0.001 when comparing TTX to the CTL condition in siROCK1 cells or in siROCK2 cells. NS stands for not statistically different. ( D ) Representative Western blotting analysis of ROCK-1 (upper panel) or ROCK-2 (lower panel) protein expression in untreated SW620 cells, in cells transfected with irrelevant siRNA (siCTL), or with siROCK1 (upper panel), or with siROCK2 (lower panel) for 48 h. β-actin was used as loading control protein. These blots are representative of four independent experiments. ( E ) Upper panel, representative fast inward sodium currents recorded in shCTL SW620 cells (left) in response to a membrane depolarizing step from − 100 to − 5 mV for 50 ms. This current was absent in SW620 cancer cells stably expressing a short hairpin RNA targeting S CN5A gene expression (shNa V 1.5 cells, right). Lower panel, effect of Y-27632 (10 µM) on cell invasiveness of SW620-sh CTL and SW620-shNa V 1.5 cancer cells. Results are expressed as ratios of mean results obtained with shCTL cells in CTL condition (vehicle). The dashed line indicates a ratio of 1. Results are from 9 independent experiments and were analysed using Mann–Whitney rank sum tests. *** P < 0.001 compared to CTL condition in shCTL cells; ++ P < 0.01 compared to Y-27632 condition in shCTL cells, ### P < 0.001 compared to the CTL condition in shNa V 1.5 cells. ( F ) Representative western blotting analysis of Na V 1.5 protein expression in shCTL and shNa V 1.5 cell lines, as compared to rat Heart Protein Extract (H.P.E) used as a positive control. HSC70 was used as loading control protein. This blot is representative of three independent experiments.

Article Snippet: For each condition, 4 × 10 5 cells were incubated with 1.6-μg anti-Na V 1.5 antibody (ASC-005, ALOMONE LABS, Israel) for 30 min at 4 °C followed by incubation with secondary antibody Alexa-Fluor-488 (1:500, A-11008, THERMO FISHER SCIENTIFIC, France) for 45 min at 4 °C.

Techniques: MANN-WHITNEY, Expressing, Transfection, Western Blot, Stable Transfection, shRNA, Positive Control

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Figure Lengend Snippet: ROCK inhibitor Y-27632 increases Na V 1.5 activity in SW620 colon cancer cells. ( A ) Representative whole-cell current recordings obtained from SW620 cells incubated with either vehicle (0.1% DMSO) as a Control (top recordings, blue traces), or 10 µM Y-27632 (bottom, red traces) for 48 h, in response to depolarizing 30-ms pulses from − 95 to + 60 mV in 5-mV steps applied every 2 s from a holding potential of − 100 mV. Dotted lines indicate baseline levels (zero current). ( B ) Sodium current density–voltage (I Na -V) relationship for Na V 1.5 channels in SW620 cells in control (0.1% DMSO, n = 23) or 10 µM Y-27632 (n = 33). Peak sodium currents were averaged and plotted as a function of the membrane voltage. ( C ) Activation- and steady-state inactivation-voltage relationships of Na V 1.5 currents recorded from SW620 cells in absence (control, black-filled circles) and presence (red-filled diamonds) of 10 µM Y-27632. Smooth lines correspond to the Boltzmann’s function fits and the V 1/2 values obtained were the following: V 1/2 -activation voltage of − 23.4 ± 0.5 mV and − 26.1 ± 0.4 mV for control and Y-27632, respectively; and V 1/2 -inactivation of − 72.2 ± 0.6 mV and − 74.5 ± 0.4 mV for control and Y-27632, respectively. ( D , E ) Peak- and persistent sodium current densities evoked from a depolarizing step from -100 to − 5 mV in absence (black bars) and presence of 10 µM Y-27632 (red bars) obtained from the same cells than in B. * P < 0.05 and ** P < 0.01 (Mann Whitney rank sum test).

Article Snippet: For each condition, 4 × 10 5 cells were incubated with 1.6-μg anti-Na V 1.5 antibody (ASC-005, ALOMONE LABS, Israel) for 30 min at 4 °C followed by incubation with secondary antibody Alexa-Fluor-488 (1:500, A-11008, THERMO FISHER SCIENTIFIC, France) for 45 min at 4 °C.

Techniques: Activity Assay, Incubation, Activation Assay, MANN-WHITNEY