Fig 1: Confocal microscopy images of HEK293T cells expressing wild-type YFP-ClC-1 channel and YFP-ClC-1 G411C mutant. (A) Representative confocal single optical plane image showing localization of wild-type YFP-ClC-1 (green signal), plasma membrane marker WGA-555 (red signal), and colocalization (yellow signal) in a dividing cell doublet. (B) Representative dividing cell doublet showing localization of YFP-G411C (green signal), plasmalemma WGA-555 (red signal), and colocalization (yellow signal). (C,D) Scatter-plots of pixel intensity in images (A,B), showing the relationship between WT or G411C and WGA. Linear regressions (black lines) demonstrate significant colocalization of WT hClC-1 with WGA [Pearson's correlation coefficient (P) of 0.7] and anticolocalization of G411C with WGA (P = 0.4). (E) Histograms reporting the Pearson's coefficient (P) of pixel intensity scatter-plot linear regressions (as in C,D), and the Mander's overlap coefficients (M1 and M2) of the two markers (WGA-555 and YFP-ClC-1). M1 indicates summed intensities of red pixels overlapping with green to the total red intensity (proportion of WGA overlapping with ClC-1). M2 indicates summed intensities of green pixels overlapping with red to the total green intensity (proportion of ClC-1 overlapping with WGA). Each bar is the mean ± SD from 64 (WT) and 34 (G411C) cell clusters. *p < 0.001 between WT and G411C hClC-1 with unpaired Student's t-test.
Fig 2: ClC-1 protein expression in HEK293T cells transfected with WT and V947E ClC-1 channels and effects of 50 µM NFA incubation for 24 h. (A). Representative western blot of total ClC-1 and ß-actin proteins from transfected HEK293T with WT and V947E in the absence or presence of 50 µM NFA. (B). Quantification of ClC-1 total protein expression level. The ClC-1 signal of each column was standardized to the ß-actin signal and normalized to WT on the same blot. Each bar is the mean ± S.E.M. from three independent experiments. Asterisks denote a significant difference (p < 0.05) between V947E in the control condition and after NFA treatment. (C). Representative western blot of surface and cytoplasmic ClC-1 and ß-actin proteins obtained from the biotinylation assay of transfected HEK293T cells incubated for 24 h in the absence or presence of 50 µM NFA. (D). Quantification of surface and cytoplasmic ClC-1 protein distribution. Each bar is the mean ± SD from three independent experiments (*at least p < 0.05 vs WT in the control condition, # at least p < 0.05 vs V947E in the control condition).
Fig 3: ClC-1 protein expression in HEK293T cells transfected with WT or G411C ClC-1 channels and effects of MG132 incubation. (A) Representative western blot of total ClC-1 and ß-actin proteins from transfected HEK293T incubated for 16 h in absence or presence of 20 µM MG132. (B) Quantification of ClC-1 total protein expression level. The ClC-1 signal of each column was standardized to ß-actin signal and normalized to WT on the same blot. Each bar is the mean ± S.E.M. from three independent experiments. Asterisks denote significant difference (p < 0.04) between control condition and MG132 treatment using two-way ANOVA. (C) Representative western blot of surface and cytoplasmic ClC-1 and ß-actin proteins obtained from biotinylation assay of transfected HEK293T cells incubated for 16 h in absence or presence of 20 µM MG132. (D) Quantification of surface and cytoplasmic ClC-1 protein distribution. Each bar is the mean ± S.E.M. from 3 to 4 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Sidak's multiple comparison test (* at least p < 0.05 vs. WT and GC + MG132).
Fig 4: Probands' EMG, pedigree, and ClC-1 mutation. (A) Repetitive nerve stimulation (RNS) on abductor digiti minimi muscle. (B) Pedigrees of the Russian family affected by Becker disease. Squared symbols indicate men and round symbols indicate women. Empty symbols denote healthy individuals and dark filled symbols denote affected individuals. The arrow indicates the proband in the family. (C) Amino acid alignment of the ClC-1 chloride channel among different species and human CLC members. (D) Three-dimensional representation of hClC-1 channel modeled upon the X-ray structure of a eukaryotic Cl-/H+ exchanger CmClC showing the localization of G411C mutation.
Fig 5: Acute application of NFA on WT, A531V, and V947E ClC-1 chloride channels. (A). Representative traces of WT, A531V, and V947E chloride currents at -90 and +60 mV before (in black) and after (in red) the application of 50 µM NFA. (B). Percentage of block of instantaneous and steady-state chloride current for WT, A531V, and V947E mutants measured at -90 mV and +60 mV induced by NFA 50 µM. (C). Values of V0.5 (half-maximal activation potential) of WT, A531V, and V947E channels before and after the application of NFA. Each dot represents the V0.5 value obtained from a single recording. Data are shown as box-and-whisker plots. Median (solid line); mean (dash line); and 10th, 25th, 75th, and 90th percentiles are indicated. The number of examined cells is reported in brackets. Asterisk denotes a significant difference compared with the relative control condition (*p < 0.05).
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