Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of Aβ 1–42 exposure on Na V 1.6 protein expression and activity in primary hippocampal neurons at 10–12 DIV. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control. ( D ) Representative traces of Na + currents recorded under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. (E) Representative traces of Na + currents recorded after 5 μM Aβ 1–42 (24 h) alone, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. ( F ) Normalization of Na + current densities, at −20 mV, represented in panel D and E. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus control, # p < 0.001 versus control Aβ 1–42 . (G) Representative western blot of Na V 1.6 protein expression (top) in the presence of siNa V 1.6 (50 nM; 48 h) in primary hippocampal neurons at 12 DIV. Quantification of siNav1.6 inhibition in primary hippocampal neurons (bottom). Values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control neurons.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Expression and activity of Na V 1.6 channels in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT and Tg2576 primary hippocampal neurons after 8 and 12 DIV. ( B ) Normalization of Na + current densities at −20 mV represented in panel A. Values are expressed as mean ± SEM of current densities of 3 independent experimental sessions. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( D ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( E ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. (F) Representative confocal images displaying Na V 1.6 distribution in WT (left) and Tg2576 (right) primary hippocampal neurons after 12 DIV. Scale bars: 20 μm.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of siNa V 1.6 and anisomycin on Na V 1.6 protein expression and activity in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( B ) Representative traces of Na + currents recorded in Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( C ) Normalization of Na + current densities at −20 mV represented in panel A and B. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control WT, *** p < 0.001 versus control WT, # p < 0.001 versus control Tg2576. ( D ) Representative current tracings recorded in the gap-free mode in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( E ) Quantification of spike frequency recorded in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. # p < 0.001 versus control Tg2576. ( F ) Quantification of membrane depolarization recorded in WT and Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. # p < 0.001 versus control Tg2576

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Immunocytochemical analysis of Na V 1.6 protein expression after anisomycin treatment in Tg2576 primary hippocampal neurons at 12 DIV. ( A ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in WT (a-c) and Tg2576 primary hippocampal neurons in the absence (d-f) or in the presence (g-i) of anisomycin. Scale bars in a-i: 20 μm. ( B ) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 primary hippocampal neurons in the absence or in the presence of anisomycin. Scale bars: 5 μm. Data are expressed as mean ± SEM of values obtained from 20 cells per group in 3 independent experimental sessions. ** p < 0.01 versus WT; # p < 0.001 versus Tg2576.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Evaluation of Na V 1.6 protein expression in the hippocampus of 3-month-old WT and Tg2576 mice. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. ( D ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in the hippocampus of 3-month-old WT (a-c) and Tg2576 mice (d-f). Scale bars in a-f: 20 μm. (E) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 neurons in the hippocampus of 3-month-old mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. **p < 0.01 versus WT.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1101/2022.03.01.480798

Figure Lengend Snippet: a: Schematic representation of the NF186 structure with clickable labeling sites (red) and the C-terminal hemagglutinin (HA) tag (purple). b – c : Representative confocal images of primary rat cortical neurons at day in vitro (DIV) 11 expressing NES PylRS AF /tRNA Pyl and NF186 WT -HA or NF186 K680TAG -HA under the human neuron-specific enolase (hNSE) promoter, in the presence of the unnatural amino acid TCO*A-Lys. Prior to imaging, living neurons were click-labeled with ATTO488-tetrazine (tz), fixed, and immunostained with anti-HA and anti-ankyrin G (ankG) primary antibodies, followed by Alexa Fluor (AF) 555- and AF633-conjugated secondary antibodies. d : Distribution of the AIS lengths measured in confocal images of the anti-ankG immunostained rat neurons expressing NF186 WT -HA, NF186K519TAG-HA, NF186 K604TAG -HA, or NF186 K680TAG -HA (HA+) and the corresponding neighboring untransfected (HA-) neurons. The box plots indicate the median (the black lines inside the box), the 25 th and 75 th percentiles (the box boundaries), the single data points (the dots), and the outliers (the yellow dots). The whiskers lengths are defined by the minimum and maximum data points. The non-parametric Kruskal-Wallis test with Dunn-Bonferroni posthoc did not show any significant differences between the HA+ and HA-AIS lengths for WT and the three clickable mutants (p WT = 0.206, p K519TAG = 0.265 p K604TAG = 1.000, and p K680TAG = 0.605; number (n) of analyzed cells: n WT HA+ =18, n WT HA- = 41, n K519TAG HA+ = 11, n K519TAG HA- = 25, n K604TAG HA+ = 10, n K604TAG HA- = 9, n K680TAG HA+ = 18, and n K680TAG HA- = 23). The details of the statistical analysis are shown in Supplementary Table 1 . e : Representative confocal image of a living neuron at DIV 11 expressing NF186 K680TAG- HA labeled with ATTO488-tz. The Z-stack images are shown as maximum intensity projections in all panels. Scale bars: 10 μm (b , c , e) . The scheme in panel a was made in BioRender.com .

Article Snippet: The primary antibodies were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183); rabbit anti-HA tag (C29F4) monoclonal antibody (1:1000 for Na V 1.6 and 1:2000 for NF186; Cell Signaling, cat. no. 3724); mouse anti-ankyrin G antibody (1:50; Santa Cruz, cat. no. 12719); and mouse anti-βIII-tubulin antibody (1:1000; BioLegend, cat. no. 801202).

Techniques: Labeling, In Vitro, Expressing, Imaging

Journal: bioRxiv

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1101/2022.03.01.480798

Figure Lengend Snippet: a : Schematic representation of the Na V 1.6 alpha subunit with the clickable labeling sites (K1425TAG and K1546TAG; red stars) and the C-terminal hemagglutinin (HA) tag (purple). In addition, the scheme depicts the Y371C mutation (yellow star) that rendered Na V 1.6 tetrodotoxin (TTX)-resistant. b – d : Representative confocal images of primary rat cortical neurons at DIV 12 expressing NES PylRS AF /tRNA Pyl and Na V 1.6 WT -HA, Na V 1.6 K1425TAG -HA, or Na V 1.6 K1546TAG -HA in the presence of the UAA TCO*A-Lys. The neurons were transfected with either Lipofectamine 2000 (b,c,e) or Lipofectamine 3000 (d) reagent. Prior to imaging, living neurons were click-labeled with ATTO488-tetrazine (tz), fixed, and immunostained with an anti-HA primary antibody, followed by an Alexa Fluor 555-conjugated secondary antibody. e : Representative confocal image of a living rat neuron at DIV 12 expressing Na V 1.6 K1546TAG -HA labeled with ATTO488-tz. f : Distribution of the AIS lengths measured in confocal images of anti-ankG immunostained rat neurons expressing Na V 1.6 K1425TAG -HA or Na V 1.6 K1546TAG -HA (HA+) and the corresponding neighboring untransfected (HA-) neurons. The box plots indicate the median (the black lines inside the box), the 25 th and 75 th percentiles (the box boundaries), and the single data points (the dots). The whiskers lengths are defined by the minimum and maximum data points. One-way ANOVA with Tukey posthoc did not show significant differences between the HA+ and HA-AIS lengths for both clickable mutants (p K1425TAG = 0.124, p K1546TAG = 0.136; number (n) of analyzed cells: n K1425TAG HA+ = 18, n K1425TAG HA- = 34, n K1546TAG HA+ = 15, and n K1546TAG HA- = 22). The details of the statistical analysis are shown in Supplementary Table 2 . Representative images of rat neurons immunostained with anti-ankG and anti-HA antibodies used for the quantitative analysis are shown in panel f. All images, except for that of Na V 1.6 WT -HA (shown in panel b), were taken as Z-stacks and are shown as maximum intensity projections. For the comparison of the Lipofectamine 2000 transfection reagent and Lipofctamine 3000, the brightness and contrast of the panels showing the HA channels ( b - d ) were linearly adjusted as indicated by the look-up-table (LUT) intensity scale bar. LUT intensity scale bars show the minimum and maximum grey values. Scale bars: 10 μm ( b – f ). The scheme in panel a was made in BioRender.com .

Article Snippet: The primary antibodies were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183); rabbit anti-HA tag (C29F4) monoclonal antibody (1:1000 for Na V 1.6 and 1:2000 for NF186; Cell Signaling, cat. no. 3724); mouse anti-ankyrin G antibody (1:50; Santa Cruz, cat. no. 12719); and mouse anti-βIII-tubulin antibody (1:1000; BioLegend, cat. no. 801202).

Techniques: Labeling, Mutagenesis, Expressing, Transfection, Imaging, Comparison

Journal: bioRxiv

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1101/2022.03.01.480798

Figure Lengend Snippet: a : Representative Na + current traces obtained from N1E-115-1 cells expressing either Na V 1.6 WT, Y371C -HA (black) or Na V 1.6 K1546TAG, Y371C -HA (red). b : Representative Na + current traces obtained from N1E-115-1 β1β2 stable cells expressing either Na V 1.6 WT, Y371C -P2A-eGFP (black) or Na V 1.6 K1425TAG, Y371C -P2A-eGFP (red). c: Peak Na + current densities as normalized to cell capacitance, were plotted vs. voltage (left panel; number of analyzed (n) cells: n WT = 20 and n K1546TAG = 18), voltage-dependence of activation (middle panel; n WT = 20 and n K1546TAG = 18), and voltage-dependence of fast inactivation (right panel; n WT = 20 and n K1546TAG =17) for K1546TAG (red) vs. WT comparison (black). d : Peak Na + current densities as normalized to cell capacitance, were plotted vs. voltage (left panel; n WT = 18 and n K1425TAG = 20), voltage-dependence of activation (middle panel; n WT = 18 and n K1425TAG =20), and voltage-dependence of fast inactivation (right panel; n WT = 18 and n K1425TAG =20) for K1425TAG (red) vs. WT comparison (black). The lines represent the Boltzmann functions fit to the data points. Shown are mean ± standard errors of the mean (SEMs; c , d ). Detailed statistical analyses are provided in Supplementary Table 3 . e – g : Representative 3D direct stochastic optical (dSTORM) super-resolution images of primary rat cortical neurons at DIV 12-14 expressing NES PylRS AF /tRNA Pyl and ( e ) Na V 1.6 WT -HA, ( f ) Na V 1.6 K1425TAG -HA, or ( g ) Na V 1.6 K1546TAG -HA in the presence of UAA TCO*A-Lys. Four to six days after the transfection, the neurons were click-labeled, fixed and immunostained with an anti-HA primary antibody and an Alexa Fluor Plus 647-conjugated secondary antibody. The left panels show the TIRF/HILO images of the anti-HA channel, acquired with 647 nm laser illumination prior to the 3D dSTORM imaging. The middle panels show the corresponding 3D dSTORM images, including a magnified view of the boxed region. The z positions in the 3D dSTORM images are color-coded according to the height maps shown on the right. The height maps contain minimal and maximal z position values. Scale bars: 5 μm for the TIRF/HILO and dSTORM images and 2 μm for the magnified views of the boxed regions ( e–g ).

Article Snippet: The primary antibodies were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183); rabbit anti-HA tag (C29F4) monoclonal antibody (1:1000 for Na V 1.6 and 1:2000 for NF186; Cell Signaling, cat. no. 3724); mouse anti-ankyrin G antibody (1:50; Santa Cruz, cat. no. 12719); and mouse anti-βIII-tubulin antibody (1:1000; BioLegend, cat. no. 801202).

Techniques: Expressing, Activation Assay, Comparison, Transfection, Labeling, Imaging

Journal: bioRxiv

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1101/2022.03.01.480798

Figure Lengend Snippet: a – b : Na + current recordings of mouse Na V 1.6 pathogenic variants combined with TAG mutations in N1E-115-1 cells. a : Representative Na + current traces obtained from N1E-115-1 cells expressing Na V 1.6 K1425TAG, Y371C -HA (red), Na V 1.6 K1425TAG, Y371C, I1652N -HA (blue), or Na V 1.6 K1425TAG, Y371C, T1785P -HA (violet). Combined with the K1425TAG Y371C mutant, both the I1652N and T1785P variants significantly reduced the peak Na + current density compared to the K1425TAG Y371C mutant alone (Na V 1.6 K1425TAG, Y371C -HA: −72.9 ± 16.4 pA/pF, number of analyzed cells (n) = 11; Na V 1.6 K1425TAG, Y371C, I1652N -HA: −12.3 ± 5.0 pA/pF, n = 8, p = 0.0015; Na V 1.6 K1425TAG, Y371C, T1785P -HA: −12.7 ± 4.4 pA/pF, n = 12, p = 0.0006; ANOVA on ranks with Dunn’s posthoc test). b : Representative Na + current traces obtained from the N1E-115-1 cells expressing Na V 1.6 K1546TAG, Y371C -HA (red), Na V 1.6 K1546TAG, Y371C, I1652N -HA (blue), or Na V 1.6 K1546TAG, Y371C, T1785P -HA (violet). When combined with the K1546TAG Y371C mutant, both the I1652N and T1785P variants significantly reduced the peak Na + current density compared to the K1546TAG Y371C mutant alone (Na V 1.6 K1546TAG, Y371C -HA: −125.1 ± 34.8 pA/pF, n = 13; Na V 1.6 K1546TAG, Y371C, I1652N -HA: −18.7 ± 6.3 pA/pF, n = 8, p = 0.0018; Na V 1.6 K1546TAG, Y371C, T1785P -HA: −15.4 ± 2.0 pA/pF, n = 8, p=0.0014; ANOVA on ranks with Dunn’s posthoc test). Shown are the mean ± standard errors of the mean (SEMs; a , b ). c : Representative confocal images of mouse hippocampal neurons at DIV 12 expressing NES PylRS AF /tRNA Pyl , Na V 1.6 K1546TAG, Y371C -HA, Na V 1.6 K1546TAG, Y371C, I1652N -HA, or Na V 1.6 K1546TAG, Y371C, T1785P -HA in the presence of the TCO*A-Lys. Four days after the transfection, the neurons were click-labeled with ATTO488-tetrazine(tz), fixed, and immunostained with an anti-HA primary antibody and an Alexa Fluor 555-conjugated secondary antibody. d : Distribution of the mean ATTO488-tz fluorescence intensity measured in confocal images of click-labeled mouse neurons expressing the control (Na V 1.6 K1546TAG, Y371C -HA) or one of the LOF variants (Na V 1.6 K1546TAG, Y371C, I1652N -HA or Na V 1.6 K1546TAG, Y371C, T1785P -HA). The box plots indicate the median (the black lines inside the box), the 25 th and 75 th percentiles (the box boundaries), the single data points (the dots), and the outliers (the yellow dots). The whiskers lengths are defined by the minimum and maximum data points. The non-parametric Kruskal-Wallis test did not show any significant differences between the control and the LOFs (p = 0.209, n K1546TAG Y371C = 20, n K1546TAG Y371C I1652N = 17 and n K1546TAG Y371C T1785P = 19). Detailed statistical analyses are provided in Supplementary Table 4 . The Z-stack images are shown as maximum intensity projections. Scale bars: 10 μm ( c ).

Article Snippet: The primary antibodies were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183); rabbit anti-HA tag (C29F4) monoclonal antibody (1:1000 for Na V 1.6 and 1:2000 for NF186; Cell Signaling, cat. no. 3724); mouse anti-ankyrin G antibody (1:50; Santa Cruz, cat. no. 12719); and mouse anti-βIII-tubulin antibody (1:1000; BioLegend, cat. no. 801202).

Techniques: Expressing, Mutagenesis, Transfection, Labeling, Fluorescence, Control

Journal: bioRxiv

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1101/2022.03.01.480798

Figure Lengend Snippet: a: Schematic representation of a combination of plasmid transfection and transduction with AAV9A2 viral vectors in primary rat cortical neurons. b – c : Representative confocal images of neurons at DIV 11 expressing NES PylRS AF /4xtRNA Pyl and ( b ) Na V 1.6 K1425TAG -HA or ( c ) Na V 1.6 K1546TAG -HA in the presence of UAA TCOA*-Lys. The neurons were transfected with clickable Na V 1.6 and transduced with orthogonal translational machinery components: NES PylRS AF (AAV#1) and four copies of tRNA Pyl (AAV#2) at DIV 8. Prior to imaging, living neurons were click-labeled with ATTO488-tetrazine (tz), fixed, and immunostained with an anti-HA primary antibody, followed by an Alexa Fluor 555 secondary antibody. The Z-stack images are shown as maximum intensity projections. For comparison with the transfected neurons (as shown in ), the brightness and contrast of the panels showing the HA channel were linearly adjusted to show the same display range (0–20,000). In addition, they were adjusted to show a broader display range (0–50,000), as indicated by the look-up-table (LUT) intensity scale bar. LUT intensity scale bars show the minimum and maximum grey values. Scale bars: 10μm ( b – c ).

Article Snippet: The primary antibodies were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183); rabbit anti-HA tag (C29F4) monoclonal antibody (1:1000 for Na V 1.6 and 1:2000 for NF186; Cell Signaling, cat. no. 3724); mouse anti-ankyrin G antibody (1:50; Santa Cruz, cat. no. 12719); and mouse anti-βIII-tubulin antibody (1:1000; BioLegend, cat. no. 801202).

Techniques: Plasmid Preparation, Transfection, Transduction, Expressing, Imaging, Labeling, Comparison

Journal: Journal of Cell Science

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1242/jcs.260600

Figure Lengend Snippet: Genetic code expansion and click labeling of NF186. (A) Schematic illustration of the NF186 structure with clickable labeling sites (red) and the C-terminal HA tag. The scheme was created with BioRender.com. (B) Western blot analysis of lysates of ND7/23 cells co-expressing NES PylRS AF /tRNA Pyl and the CMV-NF186–HA constructs indicated. Recombinant NF186–HA was detected with an anti-HA antibody. β3-tubulin was used as a loading control. (C,D) Confocal images of rat neurons at days in vitro (DIV) 11 expressing the plasmids indicated. Before imaging, living neurons were labeled with ATTO488-tz, fixed, and immunostained with anti-HA and anti-ankG antibodies. (E) Distribution of the AIS lengths measured in confocal images of the anti-ankG-immunostained neurons expressing the indicated hNSE-NF186–HA plasmids (HA+) and the neighboring untransfected (HA−) neurons. No significant differences between the groups were detected [ P >0.05, Kruskal–Wallis test followed by Dunn post-hoc analysis with Bonferroni correction for multiple comparisons; number ( n ) of analyzed neurons: n WT HA+ =18, n WT HA− =41, n K519TAG HA+ =11, n K519TAG HA− =25, n K604TAG HA+ =10, n K604TAG HA− =9, n K680TAG HA+ =18, n K680TAG HA− =23]. (F) Distribution of the mean ATTO488-tz and anti-HA fluorescence intensities (FI), and anti-HA/ATTO488-tz ratio measured in confocal images of neurons expressing the hNSE-NF186–HA constructs indicated. No significant differences between the groups were detected [ P >0.05, Kruskal–Wallis test; n WT =10, n K519TAG =16, n K604TAG =14, n K680TAG =15 neurons]. (G) Representative confocal image of an ATTO488-tz-labeled living neuron at DIV 11 expressing NF186 K680TAG –HA. (H,I) Representative 3D dSTORM images of rat neurons at DIV 12 expressing the indicated constructs and click labeled with AF647-pyr-tz or immunostained with the antibodies indicated. The z -positions are color-coded according to the height maps (bottom left). The height maps show minimal and maximal z position values. Boxed regions from H with the representative overlaid line ROIs that were used for autocorrelation analysis are shown in I. (J) The averaged autocorrelation curves from 1-µm-long intensity profiles along AISs expressing the constructs indicated ( n panNF =20, n HA-AF647 =21, n AF647-pyr-tz =21 individual ROIs measured from n panNF =8, n HA-AF647 =10, n AF647-pyr-tz =11 neurons). Mean±s.e.m. spacing values ( s ) are as follows: s panNF =170.1±23.5, s HA-AF647 =168.8±19.3, s AF647-pyr-tz =174.3±28.5. (K,L) Distribution of spacing (K) and autocorrelation amplitude (L) values of individual ROIs for the conditions indicated. No significant differences between the groups were detected [ P >0.05, one-way ANOVA with Tukey's post-hoc analysis (K) or Kruskal–Wallis test (L); n panNF =20, n HA− AF647 =21, n AF647-pyr-tz =21 individual ROIs measured from n panNF =8, n HA-AF647 =10, n AF647-pyr-tz =11 neurons]. All box plots indicate the median (the black lines inside the box), the 25th and 75th percentiles (the box boundaries), single data points (dots), outliers (yellow dots), and the furthest outliers (yellow squares). Whisker lengths are defined by the minimum and maximum data points. The Z -stack confocal images are shown as maximum intensity projections in all panels. The brightness and contrast of the panels were linearly adjusted as indicated by the look-up-table (LUT) intensity scale bar. The LUT intensity scale bars show the minimum and maximum gray values. Dots represent individual neurons (E,F) or axonal ROIs (K,L). Data were collected in two (J, K, L), three (F) or four (E) independent experiments. Details of the statistical analysis are given in Tables S1–S6 . Scale bars: 10 µm (C, D, G), 5 µm (H), 1 µm (I). ′ab, primary antibody; ″ab, secondary antibody.

Article Snippet: The primary antibodies used were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183) was used for click labeling of NF186–HA in ND7/23 cell line; rabbit anti-HA tag (C29F4) monoclonal antibody [1:1000 for click labeling of Na V 1.6 (except for experiments involving quantification of dSTORM in which it was used at 1:250 dilution) and 1:2000 for click labeling of NF186 in primary neurons; Cell Signaling Technology, cat. no. 3724]; rabbit anti-HA tag (SG77) polyclonal antibody (1:1000 for western blot analysis; Thermo Fisher Scientific, cat. no. 71-5500); mouse anti-ankyrin G antibody (1:50; Santa Cruz Biotechnology, cat. no. 12719); mouse anti-ankyrin G (N106/36) monoclonal antibody (1:100; Neuromab, cat. no. 75-146); rabbit anti-panNF antibody (1:200; Abcam, cat. no. ab31457); mouse anti-panNa V (K58/35) monoclonal antibody (1:100; Sigma-Aldrich, cat. no. S8809); mouse anti-βIII-tubulin monoclonal antibody (1:1000; BioLegend, cat. no. 801202); AF647-conjugated anti-HA tag (C29F4) rabbit antibody (1:50 for Na V 1.6 and 1:100 for NF186; Cell Signaling Technology, cat. no. 37297).

Techniques: Labeling, Western Blot, Expressing, Construct, Recombinant, Control, In Vitro, Imaging, Fluorescence, Whisker Assay

Journal: Journal of Cell Science

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1242/jcs.260600

Figure Lengend Snippet: Genetic code expansion and click labeling of Na V 1.6 in primary neurons. (A) Schematic representation of the Na V 1.6 α subunit with the clickable labeling sites (red stars) and the C-terminal HA tag. The scheme was created with BioRender.com. (B,C) Representative confocal images of rat neurons at DIV 12 expressing the constructs indicated. Prior to imaging, living neurons were labeled with ATTO488-tz, fixed, and immunostained with an anti-HA antibody. (D) Distribution of the mean anti-HA or ATTO488-tz fluorescence intensity (FI), and the anti-HA/ATTO488-tz ratio measured in confocal images of rat neurons expressing the Na V 1.6–HA constructs indicated. Significant differences are indicated (* P <0.05, Kruskal–Wallis or Mann–Whitney U test; n WT =22, n K1425TAG =12, n K1546TAG =17 neurons). (E,F) Distribution of the mean ATTO488-tz or anti-HA FI measured in confocal images of neurons expressing Na V 1.6 K1425TAG –HA (E) or Na V 1.6 K1546TAG –HA (F) transfected using Lipofectamine 2000 or Lipofectamine 3000. No significant differences between the groups were detected ( P >0.05, Mann–Whitney U test; n K1425TAG-Lipo2K =14, n K1425TAG-Lipo3K =10, n K1546TAG-Lipo2K =8, n K1546TAG-Lipo3K =11 neurons). All box plots indicate the median (the black lines inside the box), the 25th and 75th percentiles (the box boundaries), the single data points (dots), the outliers (yellow dots) and the furthest outliers (yellow squares). Whisker lengths are defined by the minimum and maximum data points. (G) Representative confocal image of click-labeled living neuron at DIV 12 expressing Na V 1.6 K1546TAG –HA. All images were taken as Z -stacks and are shown as maximum intensity projections. The brightness and contrast of the panels were linearly adjusted as indicated by the LUT intensity scale bar. The LUT intensity scale bars show the minimum and maximum gray values. Dots represent individual neurons (D–F). Data were collected in three independent experiments. Details of the statistical analysis are given in Tables S7–S13 . Scale bars: 10 µm (B,C,G). ′ab, primary antibody.

Article Snippet: The primary antibodies used were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183) was used for click labeling of NF186–HA in ND7/23 cell line; rabbit anti-HA tag (C29F4) monoclonal antibody [1:1000 for click labeling of Na V 1.6 (except for experiments involving quantification of dSTORM in which it was used at 1:250 dilution) and 1:2000 for click labeling of NF186 in primary neurons; Cell Signaling Technology, cat. no. 3724]; rabbit anti-HA tag (SG77) polyclonal antibody (1:1000 for western blot analysis; Thermo Fisher Scientific, cat. no. 71-5500); mouse anti-ankyrin G antibody (1:50; Santa Cruz Biotechnology, cat. no. 12719); mouse anti-ankyrin G (N106/36) monoclonal antibody (1:100; Neuromab, cat. no. 75-146); rabbit anti-panNF antibody (1:200; Abcam, cat. no. ab31457); mouse anti-panNa V (K58/35) monoclonal antibody (1:100; Sigma-Aldrich, cat. no. S8809); mouse anti-βIII-tubulin monoclonal antibody (1:1000; BioLegend, cat. no. 801202); AF647-conjugated anti-HA tag (C29F4) rabbit antibody (1:50 for Na V 1.6 and 1:100 for NF186; Cell Signaling Technology, cat. no. 37297).

Techniques: Labeling, Expressing, Construct, Imaging, Fluorescence, MANN-WHITNEY, Transfection, Whisker Assay

Journal: Journal of Cell Science

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1242/jcs.260600

Figure Lengend Snippet: Characterization of the expression and functionality of Na V 1.6 TAG –HA. (A) Distribution of the AIS lengths measured in confocal images of anti-ankG-immunostained rat neurons expressing the indicated Na V 1.6–HA constructs (HA+) and the corresponding neighboring untransfected (HA−) neurons. The box plots indicate the median (the black lines inside the box), the 25th and 75th percentiles (the box boundaries), the single data points (dots), and the outliers (yellow dots). Whisker lengths are defined by the minimum and maximum data points. No significant differences between the groups were detected ( P >0.05, Kruskal–Wallis test; n WT HA+ =16, n WT HA− =20, n K1425TAG HA+ =13, n K1425TAG HA− = 21, n K1546TAG HA+ =21, n K1546TAG HA− =26 neurons). Dots represent individual neurons. Data were collected from three independent experiments. Details of the statistical analysis are given in Table S14 . (B) Representative images from three repeats of rat neurons immunostained with anti-ankG and anti-HA antibodies used for the quantitative analysis in A. All images are shown as maximum intensity projections. Scale bars: 10 µm. ′ab, primary antibody. (C) Representative Na + current traces obtained from N1E-115-1 cells expressing either Na V 1.6 WT,Y371C –HA (black) or Na V 1.6 K1546TAG,Y371C –HA (red). (D) Representative Na + current traces obtained from N1E-115-1 β1β2 stable cells expressing either Na V 1.6 WT, Y371C –P2A–eGFP (black) or Na V 1.6 K1425TAG,Y371C –P2A–eGFP (red). (E) Peak Na + current densities as normalized to cell capacitance are plotted versus voltage (left; n WT =20, n K1546TAG =18 cells), voltage-dependence of activation (middle; n WT =20, n K1546TAG =18 cells) and voltage-dependence of fast inactivation (right; n WT =20, n K1546TAG =17 cells) for K1546TAG (red) versus WT comparison (black). Compared to WT channels K1546TAG amber mutation caused a significant ( P <0.05) depolarizing shift of voltage-dependent fast inactivation. (F) Peak Na + current densities as normalized to cell capacitance are plotted versus voltage (left; n WT =18, n K1425TAG =20 cells), voltage-dependence of activation (middle; n WT =18, n K1425TAG =20 cells) and voltage-dependence of fast inactivation (right; n WT =18, n K1425TAG =20 cells) for K1425TAG (red) versus WT comparison (black). Compared to WT channels, K1425TAG amber mutation caused a significant ( P <0.05) current density reduction compared to the WT channels. The data shown in E, F were analyzed with an unpaired two-tailed Student's t -test or Mann–Whitney U test. The lines represent the Boltzmann functions fit to the data points. Shown are mean±s.e.m. (E,F). Details of the statistical analysis are given in Table S15 . Data were recorded in six (E) or five (F) independent experiments.

Article Snippet: The primary antibodies used were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183) was used for click labeling of NF186–HA in ND7/23 cell line; rabbit anti-HA tag (C29F4) monoclonal antibody [1:1000 for click labeling of Na V 1.6 (except for experiments involving quantification of dSTORM in which it was used at 1:250 dilution) and 1:2000 for click labeling of NF186 in primary neurons; Cell Signaling Technology, cat. no. 3724]; rabbit anti-HA tag (SG77) polyclonal antibody (1:1000 for western blot analysis; Thermo Fisher Scientific, cat. no. 71-5500); mouse anti-ankyrin G antibody (1:50; Santa Cruz Biotechnology, cat. no. 12719); mouse anti-ankyrin G (N106/36) monoclonal antibody (1:100; Neuromab, cat. no. 75-146); rabbit anti-panNF antibody (1:200; Abcam, cat. no. ab31457); mouse anti-panNa V (K58/35) monoclonal antibody (1:100; Sigma-Aldrich, cat. no. S8809); mouse anti-βIII-tubulin monoclonal antibody (1:1000; BioLegend, cat. no. 801202); AF647-conjugated anti-HA tag (C29F4) rabbit antibody (1:50 for Na V 1.6 and 1:100 for NF186; Cell Signaling Technology, cat. no. 37297).

Techniques: Expressing, Construct, Whisker Assay, Activation Assay, Comparison, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1242/jcs.260600

Figure Lengend Snippet: Click labeling allowed a localization study on the epilepsy-causing Na V 1.6 variants with loss-of-function effect. (A,B) Na + current recordings of the indicated clickable mNa V 1.6 pathogenic variants in N1E-115-1 cells. (A) Representative Na + current traces obtained from N1E-115-1 cells expressing Na V 1.6 K1425TAG, Y371C –HA (red), Na V 1.6 K1425TAG,Y371C,I1652N –HA (blue), or Na V 1.6 K1425TAG,Y371C,T1785P –HA (violet). Combined with the K1425TAG Y371C mutant, both the I1652N and T1785P variants significantly reduced the peak Na + current density compared to the K1425TAG Y371C mutant alone (Na V 1.6 K1425TAG, Y371C –HA: −72.9±16.4 pA/pF, n =11; Na V 1.6 K1425TAG, Y371C, I1652N –HA: −12.3±5.0 pA/pF, n =8, P =0.0015; Na V 1.6 K1425TAG,Y371C,T1785P –HA: −12.7±4.4 pA/pF, n =12, P =0.0006; ANOVA on ranks with Dunn's post-hoc test). (B) Representative Na + current traces obtained from N1E-115-1 cells expressing Na V 1.6 K1546TAG,Y371C –HA (red), Na V 1.6 K1546TAG, Y371C, I1652N –HA (blue) or Na V 1.6 K1546TAG,Y371C,T1785P –HA (violet). When combined with the K1546TAG Y371C mutant, both the I1652N and T1785P variants significantly reduced the peak Na + current density compared to the K1546TAG Y371C mutant alone (Na V 1.6 K1546TAG,Y371C –HA: −125.1±34.8 pA/pF, n =13; Na V 1.6 K1546TAG,Y371C,I1652N –HA: −18.7±6.3 pA/pF, n =8, P =0.0018; Na V 1.6 K1546TAG,Y371C,T1785P –HA: −15.4±2.0 pA/pF, n =8, P =0.0014; ANOVA on ranks with Dunn's post-hoc test). Shown are the mean±s.e.m. (C) Representative confocal images of mouse hippocampal neurons at DIV 12 co-expressing NES PylRS AF /tRNA Pyl and the mNa V 1.6 variants indicated. The neurons were labeled with ATTO488-tz, fixed, and immunostained with an anti-HA antibody. The Z -stack images are shown as maximum intensity projections. Scale bars: 10 µm. ′ab, primary antibody. (D) Distribution of the mean ATTO488-tz fluorescence intensity (FI) measured in confocal images of click-labeled neurons expressing the indicated mNa V 1.6 control or LOF variants. The box plots indicate the median (black lines inside the box), the 25th and 75th percentiles (the box boundaries), the single data points (dots), and the outliers (yellow dots). Whisker lengths are defined by the minimum and maximum data points. No significant differences between the groups were detected ( P >0.05; Kruskal–Wallis test; n K1546TAG Y371C =29, n K1546TAG Y371C I1652N =24, and n K1546TAG Y371C T1785P =26 neurons). Details of the statistical analysis in D are given in Table S16 . Data were collected from three (A) or four (B–D) independent experiments.

Article Snippet: The primary antibodies used were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183) was used for click labeling of NF186–HA in ND7/23 cell line; rabbit anti-HA tag (C29F4) monoclonal antibody [1:1000 for click labeling of Na V 1.6 (except for experiments involving quantification of dSTORM in which it was used at 1:250 dilution) and 1:2000 for click labeling of NF186 in primary neurons; Cell Signaling Technology, cat. no. 3724]; rabbit anti-HA tag (SG77) polyclonal antibody (1:1000 for western blot analysis; Thermo Fisher Scientific, cat. no. 71-5500); mouse anti-ankyrin G antibody (1:50; Santa Cruz Biotechnology, cat. no. 12719); mouse anti-ankyrin G (N106/36) monoclonal antibody (1:100; Neuromab, cat. no. 75-146); rabbit anti-panNF antibody (1:200; Abcam, cat. no. ab31457); mouse anti-panNa V (K58/35) monoclonal antibody (1:100; Sigma-Aldrich, cat. no. S8809); mouse anti-βIII-tubulin monoclonal antibody (1:1000; BioLegend, cat. no. 801202); AF647-conjugated anti-HA tag (C29F4) rabbit antibody (1:50 for Na V 1.6 and 1:100 for NF186; Cell Signaling Technology, cat. no. 37297).

Techniques: Labeling, Expressing, Mutagenesis, Fluorescence, Control, Whisker Assay

Journal: Journal of Cell Science

Article Title: Direct fluorescent labeling of NF186 and Na V 1.6 in living primary neurons using bioorthogonal click chemistry

doi: 10.1242/jcs.260600

Figure Lengend Snippet: AAV-based vectors are efficient tools for delivering orthogonal translational machinery for click labeling of Na V 1.6 in primary neurons. (A) Schematic representation of AAV9A2 viral vectors and Na V 1.6–HA plasmid used for genetic code expansion and click labeling of Na V 1.6. In addition to transfections with Na V 1.6–HA constructs, neurons were either transfected with NES PylRS AF /tRNA Pyl or transduced with orthogonal translational machinery components (AAV#1, AAV#2 and AAV#7). The schemes were created with BioRender.com. (B) Representative confocal images of neurons at DIV 11 expressing the constructs indicated. Prior to imaging, living neurons were labeled with ATTO488-tz, fixed, and immunostained with an anti-HA antibody. The Z -stack images are shown as maximum intensity projections. The brightness and contrast of the panels were linearly adjusted as indicated by the LUT intensity scale bar. LUT intensity scale bars show the minimum and maximum gray values. Scale bars: 10 µm. ′ab, primary antibody. (C) Distribution of the mean ATTO488-tz or anti-HA fluorescence intensity (FI) measured in confocal images of transfected or transduced neurons. Significant differences are indicated (* P <0.05, Mann–Whitney U test; n K1425TAG-1 =30, n K1546TAG-1 =35, n K1425TAG-2 =28, n K1546TAG-2 =38, n K1425TAG-3 =22, n K1546TAG-3 =28, n K1425TAG-4 =29, n K1546TAG-4 =29 neurons). All box plots indicate the median (black lines inside the box), the 25th and 75th percentiles (box boundaries), the single data points (dots), the outliers (yellow dots) and the furthest outliers (yellow squares). Whisker lengths are defined by the minimum and maximum data points. Dots represent individual neurons. Details of the statistical analysis are given in Tables S17 – . Data were collected from three independent experiments.

Article Snippet: The primary antibodies used were as follows: mouse anti-HA tag (2-2.2.14) antibody (1:1000; Thermo Fisher Scientific, cat. no. 26183) was used for click labeling of NF186–HA in ND7/23 cell line; rabbit anti-HA tag (C29F4) monoclonal antibody [1:1000 for click labeling of Na V 1.6 (except for experiments involving quantification of dSTORM in which it was used at 1:250 dilution) and 1:2000 for click labeling of NF186 in primary neurons; Cell Signaling Technology, cat. no. 3724]; rabbit anti-HA tag (SG77) polyclonal antibody (1:1000 for western blot analysis; Thermo Fisher Scientific, cat. no. 71-5500); mouse anti-ankyrin G antibody (1:50; Santa Cruz Biotechnology, cat. no. 12719); mouse anti-ankyrin G (N106/36) monoclonal antibody (1:100; Neuromab, cat. no. 75-146); rabbit anti-panNF antibody (1:200; Abcam, cat. no. ab31457); mouse anti-panNa V (K58/35) monoclonal antibody (1:100; Sigma-Aldrich, cat. no. S8809); mouse anti-βIII-tubulin monoclonal antibody (1:1000; BioLegend, cat. no. 801202); AF647-conjugated anti-HA tag (C29F4) rabbit antibody (1:50 for Na V 1.6 and 1:100 for NF186; Cell Signaling Technology, cat. no. 37297).

Techniques: Labeling, Plasmid Preparation, Transfection, Construct, Transduction, Expressing, Imaging, Fluorescence, MANN-WHITNEY, Whisker Assay

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Exploration of SCN8A expression in the neoplasia-carcinoma sequence of human cervical tissue. ( A ) Expression levels of SCN8A gene in low-grade (CIN1, n = 23) and high-grade cervical intraepithelial neoplasia (CIN2/3, n = 16), as well as in invasive cervical cancer positive to HPV16 (HPV16-CeCa, n = 35) and invasive cervical cancer positive to other oncogenic HPV types (HPV-CeCa, n = 22), versus non-cancerous cervix (NCC, n = 20). Bars show the average fold-change ratio (2 −ΔΔCt ) of SCN8A gene for individual samples of each group. ( B ) Scattering plot of fold-change values for each group of samples. Horizontal black lines represent the fold-change mean values. SCN8A gene was significantly upregulated in cervical cancer tissues (Mann-Whitney U test, P < 0.0001). ( C ) Western blot analysis of Na V 1.6 channel expression in total protein extracts from NCC, CIN and CeCa samples. Total protein extracts from HEK293 cells stably expressing Na V 1.6 channels were used as positive control. Representative results of four independent experiments. Numbers below blots correspond to sample number. Samples 266 and 275 are those that were present in all western blots experiments shown in the present work. ( D ) Relative expression of Na V 1.6 protein in cervical tissue samples. Blots were quantified by densitometry and normalized to that of GAPDH for NCC ( n = 12), CIN ( n = 10) and CeCa ( n = 29). Asterisks indicates P < 0.05 with a Student’s t-test.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

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

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Immunohistochemical analysis of Na V 1.6 channels in human cervical tissue. ( A ) Representative images of non-cancerous cervix (NCC), low-grade cervical intraepithelial neoplasia (CIN1), high-grade cervical intraepithelial neoplasia (CIN3) and invasive cervical cancer (CeCa), showing H&E staining (left column); immunohistochemical detection of Na V 1.6 channel at ×10 and ×40 amplifications (middle columns); and negative controls (right column), where a non-specific IgG primary antibody was used in parallel slides. Scale bars represent 100 μm, and 300 μm for ×40 amplifications, respectively. ( B ) Fraction of total staining for each sample analyzed. Immunoreactivity intensity was evaluated as no signal (NS, black), Low (orange) and high (red) Na V 1.6 staining in NCC, CIN1, CIN3 and CeCa tissue samples. The number of analyzed slides is indicated in parenthesis at the top of each column.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Expression of Na V 1.6 channels in human cervical cancer cell lines. ( A ) Immunofluorescence confocal microscopy analysis of Na V 1.6 channels expression in C33A, SiHa, HeLa, and HEK293 cells stably expressing Na V 1.6 channels (HEK-Nav1.6; positive control). Cervical cancer cells were incubated with an antibody against Na V 1.6 protein followed by a staining with FITC-coupled secondary antibody. DAPI reagent was used for nucleus staining. Image acquisition was performed each 0.33 µm in a total thickness of 6.5 µm. Confocal sections were merged and 3D-reconstructions were performed from Z-planes for each region of interest. Orthogonal projections from xz and yz planes of confocal images show positive signal for Na V 1.6 protein ( far right panel ), indicated by red arrows in xz and yellow arrows in yz planes, respectively. Scale bar, 10 µm. ( B ) Western blot analysis of Na V 1.6 channel protein in total (T), cytoplasmic (C) and nuclear (N) protein extracts from human cervical cancer cell lines. Histone-3 and GAPDH proteins were used to demonstrate the enriching of subcellular fractions. Representative blot of three independent experiments. Notice that a ~150 kDa anti-Na V 1.6 reactive protein (red arrows) was found in nuclear fraction from cancer cells. The full-length ~250 kDa Na V 1.6 protein was only observed in total and cytoplasmic protein extract from C33A cells.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Expressing, Immunofluorescence, Confocal Microscopy, Stable Transfection, Positive Control, Incubation, Staining, Western Blot

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: The heterologous expression of Na V 1.6 channels boost the invasive capacity of cervical cancer cell lines. ( A ) Representative images of phase contrast and fluorescent microscopy of C33A cells 36 h after co-transfection with Na V 1.6 and GFP cDNAs. GFP-fluorescence indicated that 50–60% cancer cells were positively transfected. ( B ) Representative families of sodium currents obtained from non-transfected (black traces) and transfected C33A cells (red traces) with the Na V 1.6 channel in response to 16-ms pulses that depolarized the cell membrane from −80 to +80 mV in 10-mV steps applied every 10 s from a holding potential of −100 mV. Dotted lines indicate the baseline (zero current). Shown recordings are the average of two current traces at any given membrane potential and filtered at 5 kHz. ( C ) Current-voltage relationship for Na V 1.6 channels heterologously expressed in C33A cells. Peak Na + currents were averaged and plotted as a function of the depolarizing potential ( V m ). ( D ) Activation of normalized Na + conductance. Same cells as in ( C ). Smooth line is the fit to a Boltzmann function (see Methods) with the following parameters: V 1/2 = −12.2 ± 1.2 mV and k = 9.7 ± 1.0 mV; n = 9 cells. ( E ) The heterologous expression of Na V 1.6 channels enhances the invasive capacity of C33A cells. Relative invasion of C33A cells transfected with Na V 1.6 in absence or presence of 1 µM TTX, with respect to the control, untransfected C33A cells (black column). Columns represent the mean value of three independent experiments performed in triplicate (mean ± SD). *Statistically different from control condition ( P < 0.05).

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Expressing, Microscopy, Cotransfection, Fluorescence, Transfection, Activation Assay

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Na V 1.6 channels activity induces secretion of MMP-2 in cervical cancer cell lines. ( A ) Gelatin zymography for conditioned medium from cervical cancer cell lines. C33A, SiHa and HeLa cells were transfected with Na v 1.6 and grown for 24 h in absence or presence of 1 µM TTX. Conditioned medium was obtained and cells were lysed. Activity for gelatinases MMP-2 and MMP-9 was analyzed on equal volumes of concentrated conditioned medium by using gelatin-substrate polyacrylamide gel electrophoresis followed by an incubation in activity buffer and a staining with Coomassie blue. The conditioned medium obtained from MCF-7 cells treated with 100 ng/ml phorbol 12, 13-dibutyrate (PDB) for 40 h, was used as positive control. Proteolytic activity was detected as clear bands against a dark background of undigested substrate ( upper panel ). Total protein extracts from cell lysates were analyzed by western blotting with anti-GAPDH antibody ( bottom panel ). The results shown are representative of three independent experiments. ( B ) Secretion of MMP-2 was quantified by densitometry analysis using GAPDH bands for normalizing. Results are given as the amount of gelatin degradation showed as clear bands relative to GAPDH bands for each condition. Columns are means ± SD from three independent experiments. *Statistically significant as P < 0.05. ( C ) Representative western blot for MMP-2 expression in total protein extracts from human biopsies of NCC and CeCa. Blots were stripped and re-probed for total GAPDH as the loading control. ( D ) Expression of MMP-2 protein was studied by densitometry analysis of western blot experiments. Results are given as the amount of MMP-2 protein relative to that of GAPDH for NCC ( n = 13) and CeCa ( n = 14). Columns are means ± SEM. * Statistically significant as P < 0.05. ( E ) Western blot analysis of NHE-1 expression and ( F ) relative levels of NHE-1 protein in the same samples of panel (C) and (D), respectively. Data are means ± SEM, * P < 0.05. Samples used in ( C ) and ( E ) are exactly the same.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Activity Assay, Zymography, Transfection, Polyacrylamide Gel Electrophoresis, Incubation, Staining, Positive Control, Western Blot, Expressing

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P < 0.05 for * and P < 0.01 for ** (Mann-Whitney Rank Sum test). ( B ) Representative western blotting experiment for the analysis of pro- and mature MMP-2 forms in supernatants of C33A cultures. Conditioned medium from C33A cells transfected with Na V 1.6 and grown in the absence or the presence of 1 μM TTX was recovered after 48 h and concentrated by centrifugation. HSC70 was used as a loading control. Supernatants form C33A cells grown in complete medium (10% FBS) served as a positive control (+). ( C ) Evaluation of pro- and mature MMP-2 expression. Quantification was made by densitometric analysis of western blot images. Results are given as the amount of pro-MMP2 and MMP-2 protein relative to that of HSC70 in cell lysates. Columns are mean ± SD from three independent experiments. *Significantly different from Control at P < 0.01. There were no significant differences between proMMP-2 and MMP-2 forms in Na V 1.6-transfected C33A cells (solid red columns).

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Activity Assay, Transfection, Staining, MANN-WHITNEY, Western Blot, Centrifugation, Positive Control, Expressing