Journal: bioRxiv

Article Title: Scn4b Modulates Huntington’s Disease Phenotype Severity in vivo

doi: 10.64898/2026.03.08.708251

Figure Lengend Snippet: (A) Volcano plots of significantly differentially expressed genes (DEGs) in a CAG-length-dependent manner in the direct (dSPNs) and indirect (iSPNs) pathway spiny projection neurons of an HD mouse model allelic series (Q50, Q111, Q170, zQ175), as reported by translating ribosome affinity purification (TRAP) profiling. (B) Representative RNAScope in situ hybridization images showing striatal caudate nucleus SCN4B RNA (yellow) reduction across human HD grades 1-3 compared to pathologically normal (PN) caudate tissues. Scale bar, 5 µm. (C) Quantification of normalized (to PN) SCN4B puncta per cell. Data are shown as mean ± SD ( n =3 biological replicates per condition; 10 images (63x) per donor). One-sided t-test: ** p = 0.0031 (PN vs. HD1) and 0.0081 (PN vs. HD2); *** p = 0.0007 (PN vs. HD3).

Article Snippet: The GFP sequence was replaced with Scn4b sequence, amplified from the mouse Scn4b cDNA ORF plasmid (Scn4b #MR226154; Origene, Rockville, MD) for the pAAV GPR88 :: Scn4b OX construct.

Techniques: Affinity Purification, RNAscope, In Situ Hybridization

Journal: bioRxiv

Article Title: Scn4b Modulates Huntington’s Disease Phenotype Severity in vivo

doi: 10.64898/2026.03.08.708251

Figure Lengend Snippet: (A) Schematic showing the Scn4b knockdown (KD) approach in adult wild-type mice and the experimental timeline for behavioral testing. (B) Representative RNAScope in situ hybridization images of striatal tissue from mice injected with 5E12 vg/mouse of AAV PHP.eB hSyn::CRISPR/CasRx containing guides targeting Scn4b showing the reduction of Scn4b signal (yellow). FLuc denotes data from tissue with the guides targeting Firefly Luciferase, which is not present in the mouse genome. Scale bar, 25 µm (top) and 5 µm (bottom). (C) Relative quantification of Scn4b puncta count. Data are shown as mean ± SD ( n =4 mice per condition; 25 images (63x) per mouse). One-sided t-test: p = 0.0038. (D) Horizontal distance and (E) vertical time traveled as measured by open field test over a 30 min testing period. (F) Latency to fall in the rotarod test. (G) Stride length measured from paw prints from gait analysis. (H) Percentage of food pellets burrowed (by total mass) in the burrowing test. (I) Righting reflex time. Data are shown as mean ± SEM for all longitudinal time course data and mean ± SD for single-time-point comparisons ( n =8-10 mice per group). One-sided t-test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (J) Schematic showing the Scn4b knockout (KO) approach in conditional Scn4b floxed mice and the experimental timeline for behavioral testing. (K) Representative RNAScope in situ hybridization FISH images of striatal tissue from mice injected with 5E12 vg/mouse of AAV PHP.eB hSyn::GFP/iCre virus showing the reduction of Scn4b signal (yellow). Scale bar, 1 mm (left), 75 µm (middle) and 15 µm (right). (L) Relative quantification of Scn4b signal intensity. Data are shown as mean ± SD ( n = 4 mice per condition; 10 images (40x) per mouse). (M) Horizontal distance and (N) vertical time traveled as measured by open field test over a 60 min testing period. (O) Number of falls during rotarod training and (P) latency to fall in the rotarod test. (Q) Measured stride length of the paw prints from gait analysis. (R) Righting reflex time. Data are shown as mean ± SEM for all line graphs and as mean ± SD for all bar graphs ( n =13 mice per group). One-sided t-test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Figures in panels A and J were created with BioRender.

Article Snippet: The GFP sequence was replaced with Scn4b sequence, amplified from the mouse Scn4b cDNA ORF plasmid (Scn4b #MR226154; Origene, Rockville, MD) for the pAAV GPR88 :: Scn4b OX construct.

Techniques: Knockdown, RNAscope, In Situ Hybridization, Injection, CRISPR, Luciferase, Quantitative Proteomics, Knock-Out, Virus

Journal: bioRxiv

Article Title: Scn4b Modulates Huntington’s Disease Phenotype Severity in vivo

doi: 10.64898/2026.03.08.708251

Figure Lengend Snippet: (A) Schematic showing the snRNA-seq experimental workflow. (B) UMAP representing all cell types captured from striatal tissue of the Scn4b KD and control ( FLuc ) mice. (C) Back-to-back bar chart showing the number of genes that are dysregulated in the striatum of Scn4b KD versus FLuc mice, per cell type (log 2 fold change abs( z ) >= 1 and p adj. FDR < 0.05). (D) Volcano plot showing significant DEGs (versus FLuc , log 2 fold change abs( z ) >= 1 and p adj. FDR < 0.05) in striatal matrix dSPNs and (E) iSPNs. (F) GO BP pathway enrichment analysis of Scn4b KD downregulated DEGs (versus FLuc ) in matrix dSPNs and iSPNs. (G) Venn diagram showing the overlap between downregulated DEGs of Scn4b KD mice and human HD in matrix dSPNs and (H) iSPNs. Fisher’s exact test p = 1.32 × 10 -7 and p = 1.61 × 10 -14 , respectively. (I) GO BP pathway enrichment analysis of the shared downregulated DEGs in matrix dSPNs and iSPNs from Scn4b KD mice and human HD. (J) Overlap of the Scn4b KD DEGs and top hub genes from Langfelder et al., 2016. Fisher’s exact test: p = 0.0097 (M2 dSPN.Matrix), 0.0771 (M2 iSPN.Matrix), 0.0337 (M25 dSPN.Matrix). Figure in panel A created with BioRender.

Article Snippet: The GFP sequence was replaced with Scn4b sequence, amplified from the mouse Scn4b cDNA ORF plasmid (Scn4b #MR226154; Origene, Rockville, MD) for the pAAV GPR88 :: Scn4b OX construct.

Techniques: Control

Journal: bioRxiv

Article Title: Scn4b Modulates Huntington’s Disease Phenotype Severity in vivo

doi: 10.64898/2026.03.08.708251

Figure Lengend Snippet: (A) Schematic showing the Scn4b OX approach in the zQ175 HD model mice and the timeline used for behavioral testing. (B) Representative RNAScope in situ hybridization images of striatal tissue from mice injected with 1E12 vg/mouse of AAV PHP.eB GPR88 ::GFP or GPR88 :: Scn4b OX showing the reduction of Scn4b (yellow) in zQ175 vs. WT-GFP OX and the overexpression of Scn4b in only zQ175- Scn4b vs GFP OX. Scale bar, 10 µm. (C) Relative quantification of Scn4b expression across the experimental groups. Data are shown as mean ± SD ( n =3 mice per condition; 25 images (63x) per mouse). (D) Vertical time and (E) jump time parameters as measured by open field test over a 60 min testing period. (F) Latency to fall in the rotarod test. (G) Latency to cross in the beam walk test. (H) Grip strength measurement. For all tests, data are shown as mean ± SD ( n =7 mice per group). One-sided t-test for all parameters: * p ≤ 0.05, ** p ≤ 0.01. (I) Representative IF images (targeting EM48) of striatal sections from zQ175 mice injected with 1E12 vg/mouse of AAV PHP.eB GPR88 ::GFP or GPR88 :: Scn4b , showing the reduction of EM48+ mHTT aggregate signal (red) in Scn4b OX zQ175 mice. Scale bar, 20 µm. (J) Quantification of EM48+ mHTT aggregate count. Data are shown as mean ± SD ( n =4 mice per condition; 15 images (40x) per mouse). (K) Representative IF images (using the P90 antibody) of striatal sections from zQ175 mice injected with 1E12 vg/mouse of AAV PHP.eB GPR88 ::GFP or GPR88 :: Scn4b , showing the reduction of P90+ mHTT aggregate signal (red) in Scn4b OX zQ175 mice. Scale bar, 20 µm. (L) Quantification of P90+ mHTT aggregate count. Data are shown as mean ± SD ( n =3 mice per condition; 25 images (63x) per mouse). (M) Schematic representation of whole-cell clamp recording from the dorsal striatum. (N) The mean ± SEM of action potential thresholds for dorsal striatum SPNs from mice of the indicated genotypes. Figure in panel A created with BioRender.

Article Snippet: The GFP sequence was replaced with Scn4b sequence, amplified from the mouse Scn4b cDNA ORF plasmid (Scn4b #MR226154; Origene, Rockville, MD) for the pAAV GPR88 :: Scn4b OX construct.

Techniques: RNAscope, In Situ Hybridization, Injection, Over Expression, Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: Scn4b Modulates Huntington’s Disease Phenotype Severity in vivo

doi: 10.64898/2026.03.08.708251

Figure Lengend Snippet: (A) Schematic showing the snRNA-seq experimental workflow. (B) UMAP representing all cell types captured from striatal tissue of all mice included in this OX study. (C) Back-to-back bar chart showing the number genes that are differentially expressed (DEGs) in the striatum of Scn4b OX mice (zQ175- Scn4b OX vs zQ175-GFP OX; log 2 fold change abs( z ) >= 1 and p adj. FDR < 0.05), per cell type. (D) Volcano plot showing these genes in matrix dSPNs and (E) iSPNs of Scn4b OX mice. X-axis limit is set to ±0.4 for visualization purposes; genes exceeding this range include Ttr (log 2 fold change = −0.778) and Cmss1 (log 2 fold change = −0.777) in dSPNs and Cmss1 (log 2 fold change = −0.818) in iSPNs. GO BP pathway enrichment analysis showing the upregulated and downregulated pathways of matrix (F) dSPNs and (G) iSPNs in the zQ175 and Scn4b OX comparisons. (H) Volcano plots showing genes that are changed in opposite directions in the zQ175 and Scn4b OX comparisons in matrix dSPNs and (I) iSPNs. GO BP pathway enrichment analysis of (J) genes that are downregulated in the Scn4b OX comparison and upregulated in the zQ175 comparison and (K) genes that are upregulated in the Scn4b OX comparison and downregulated in the zQ175 comparison. (L) Heatmap showing log 2 fold change of selected MMR genes in the zQ175 and Scn4b OX comparisons. *indicates significance using the log 2 fold change abs( z ) >= 1 and p adj. FDR < 0.05 cutoff. Figure in panel A created with BioRender.

Article Snippet: The GFP sequence was replaced with Scn4b sequence, amplified from the mouse Scn4b cDNA ORF plasmid (Scn4b #MR226154; Origene, Rockville, MD) for the pAAV GPR88 :: Scn4b OX construct.

Techniques: Comparison

Journal: Nature immunology

Article Title: A voltage-gated sodium channel is essential for the positive selection of CD4 + T cells

doi: 10.1038/ni.2379

Figure Lengend Snippet: Expression of Scn5a - Scn4b VGSC during the course of positive selection. ( a ) Transcriptional profiling analysis of 28 differentially expressed genes upregulated by gp250–I-E k and simultaneously downregulated by MCC–I-E k in AND Rag1 −/− H-2 d DP thymocytes after 7 h stimulation. ( b, c ) Quantitative RT-PCR analysis of Scn4b mRNA expression in pre-selected AND. Rag1 −/− H-2 d DP thymocytes stimulated with gp250–I-E k and MCC–I-E k for various times of incubation ( b ) or in various cell subsets sorted by flow cytometry ( c ). ( d, e ) Quantitative RT-PCR analysis of Scn5a mRNA expression in pre-selected AND. Rag1 −/− H-2 d DP thymocytes stimulated with gp250–I-E k and MCC–I-E k for various times of incubation ( d ) or in various cell subsets sorted by flow cytometry ( e ). Results (mean ± s.d.; n = 2) are presented relative to Actb expression.

Article Snippet: The human SCN4B cDNA was purchased from Origene (RC223951).

Techniques: Expressing, Selection, Quantitative RT-PCR, Incubation, Flow Cytometry

Journal: Nature immunology

Article Title: A voltage-gated sodium channel is essential for the positive selection of CD4 + T cells

doi: 10.1038/ni.2379

Figure Lengend Snippet: A regulatory subunit SCN4B is critical in positive selection. ( a, b, c ) Left: Flow cytometry of thymocyte differentiation induced in reaggregate culture. The reaggregate cultures in a,b,c were set up as described in Fig. 3a,b,c respectively, with treatment of SCN4B-Ig or unrelated Ctrl-Ig (Ceacam4-Ig) fusion protein. Right: Quantification of percent subpopulation; each symbol represents a reaggregate culture. All P values were calculated by two-tailed Mann-Whitney test. ( a ) gp250 induced AND CD4SP positive selection with SCN4B-Ig or Ctrl-Ig (10 μg; mean ± s.d.; n = 6 in three experiments; * P = 0.0022). ( b ) Positive selection of AND CD4SP T cells induced by IFN-γ–treated ANV41.2 cells with 10 μg SCN4B-Ig or Ctrl-Ig (mean ± s.d.; n = 7 in three experiments; * P = 0.0041). ( c ) Positive selection of polyclonal pre-selected B6.K DP thymocytes with 1 μg SCN4B-Ig or Ctrl-Ig (mean ± s.d.; n = 9 in three experiments; * P = 0.0018). (d) Fura-2 Ca 2+ flux of pre-selected AND. Rag1 −/− H-2 d DP thymocytes stimulated with gp250–I-E k with SCN4B-Ig or Ctrl-Ig (1 μg). ( e ) Summary of peak and mean value of intracellular Ca 2+ concentration from 7.5 to 15 min of two experiments each involving 50 cells (* P < 0.0001; ** P < 0.0001)

Article Snippet: The human SCN4B cDNA was purchased from Origene (RC223951).

Techniques: Selection, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Concentration Assay

Journal: Nature immunology

Article Title: A voltage-gated sodium channel is essential for the positive selection of CD4 + T cells

doi: 10.1038/ni.2379

Figure Lengend Snippet: Peripheral AND CD4 + T cells acquire the ability to respond to positively selecting ligands by expression of human VGSCs. ( a ) Flow cytometry of AND hybridoma transduced with huSCN5A and huSCN4B plasmids containing IRES expression of GFP and DsRED fluorescence proteins respectively. ( b ) CD69 upregulation on huVGSC + AND hybridomas in response to gp250–I-E k stimulation. huVGSC + AND hybridomas and untransduced AND hybridomas were stimulated with plate bound gp250–I-E k or MCC–I-E k overnight. ( c ) CD69 upregulation on AND peripheral CD4 + T cells with or without the expression of huVGSC in respond to plate bound gp250–I-E k or MCC–I-E k stimulation. CD4 + peripheral T cells from AND. Rag1 −/− H-2 k mice were electroporated with human SCN5A-GFP and human SCN4B-DsRed plasmids. The post-electroporation cells were rested for 12 h, and stimulated with plate bound gp250–I-E k or MCC–I-E k for 10 h. ( d ) The bar graphs summarize the percent CD69-expressing cells in respond to gp250–I-E k stimulation ( n = 3 in two experiments; * P = 0.0139 by paired t test).

Article Snippet: The human SCN4B cDNA was purchased from Origene (RC223951).

Techniques: Expressing, Flow Cytometry, Transduction, Fluorescence, Electroporation

Journal: Cells

Article Title: The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells

doi: 10.3390/cells10071624

Figure Lengend Snippet: Na V β4 downregulation induces morphological changes in non-cancer mammary cells. ( a ) The expression level of the SCN4B gene, coding for Na V β4, was analysed from datasets from The Cancer Genome Atlas ( http://cancergenome.nih.gov , accessed on 19 December 2020), from the US National Cancer Institute, in the non-tumoral adjacent tissue (n = 178), and in the different stages of primary breast tumours: I (n = 125), IIA (n = 243), IIB (n = 115), IIIA (n = 85), IIIB (n = 10), IIIC (n = 31), IV (n = 4). For each array, data were log2-transformed and centred to the median. ***, statistically different with p < 0.001 (Mann–Whitney rank sum test) when comparing with adjacent non-tumoral tissue; *, p < 0.05 when comparing Stage I with stage IIA. ( b ) Na V β4 protein expression level was assessed by western blotting in non-cancer MCF10A human mammary epithelial cells and in human breast cancer MDA-MB-231 cells. The upper section shows a WB representative of 5 independent experiments. HSC70 immunodetection was used as a loading control. The lower section shows a quantification of Na V β4 protein expression in the two cell lines expressed relatively to that of MCF10A. *, statistically different with p < 0.05 (Mann–Whitney rank sum test). ( c ) Na V β4 protein expression level was assessed by western blotting in control MCF10A cells and in cells stably knocked down for the expression of SCN4B gene (MCF10A Crβ4). The upper section shows a WB representative of 8 independent experiments. HSC70 immunodetection was used as a loading control. The lower section shows a quantification of Na V β4 protein expression in the two cell lines expressed relatively to that of MCF10A CTL (n = 8). *, statistically different with p < 0.05 (Mann–Whitney rank sum test). ( d ) Representative images of MCF10A CTL and MCF10A Crβ4 cells in phase contrast microscopy. Scale bar, 50 µm. ( e ) Maximal cell length (n = 31 MCF10A CTL and n = 20 MCF10A Crβ4) and, in ( f ), number of intercellular contacts per cell (n = 60 MCF10A CTL and n = 57 MCF10A Crβ4), assessed from images taken as in ( d ). Cells were randomly selected from pictures, and the number of joint cells was manually counted. ***, statistically different with p < 0.001 (Student’s t -test). ( g ) MCF10A CTL and MCF10A Crβ4 cells were stained for the identification of nuclei (DAPI, blue staining) and F-actin (phalloidin-594, red staining). Scale bar, 125 µm. ( h ) Mean cell area (n = 40 MCF10A CTL and n = 40 MCF10A Crβ4) and, in ( i ), F-actin fluorescence intensity per cell surface (n = 100 MCF10A CTL and n = 100 MCF10A Crβ4) were calculated from images taken as in ( g ). ***, statistically different with p < 0.001 (Student’s t -test).

Article Snippet: A stable MCF10A cell line knocked-down for the expression of the SCN4B gene, coding for Na V β4, was generated using the CRISPR/Cas9 technique, as previously described [ ] by transfection with the SCN4B Double Nickase Plasmid (sc-411001, Santa Cruz, France).

Techniques: Expressing, Transformation Assay, MANN-WHITNEY, Western Blot, Immunodetection, Control, Stable Transfection, Microscopy, Staining, Fluorescence

Journal: Cells

Article Title: The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells

doi: 10.3390/cells10071624

Figure Lengend Snippet: Na V β4 prevents β-catenin degradation. ( a ) MCF10A CTL and MCF10A Crβ4 cysts were stained with Hoechst 33342 to visualize cell nuclei and immunostained to identify β-catenin and E-cadherin. Representative images from 6 independent experiments. Scale bar, 25 µm. ( b ) Expression of the CTNNB1 gene, coding for β-catenin, was analysed in the MCF10A CTL and MCF10A Crβ4 cells (n = 8 independent experiments). Statistically different from the MCF10A CTL cysts: ***, p < 0.001 (Mann–Whitney rank sum test). ( c ) Left, β-catenin protein expression level was assessed by western blotting in the MCF10A CTL and MCF10A Crβ4 cells. Representative WB from 6 independent experiments. Right, quantification of β-catenin protein expression in the MCF10A CTL and MCF10A Crβ4 cells (n = 6). **, statistically different with p < 0.01 (Mann–Whitney rank sum test). ( d ) β-catenin protein expression was assessed in untreated (Unt) MCF10A CTL and MCF10A Crβ4 cells or after the treatment with 10 µM MG132 for 3 h, 6 h, 12 h, or 24 h, or with the solvent DMSO at corresponding times. β-actin immunodetection was used as a loading control. ( e ) Quantification of β-catenin protein expression in the same conditions as in ( d ), from 5 independent experiments. Individual results are shown, centred by medians. Statistically different with p < 0.001 (two-way ANOVA). ( f ) Na V β4 protein expression was assessed by western blotting in MCF10A transfected with control “irrelevant” siRNA (siCTL) or with SCN4B -specific siRNA (siβ4) at 5 and 30 nM. ( g ) β-catenin protein expression was assessed in untreated MCF10A cells or in cells transfected with siCTL or siβ4 (30 nM). β-actin immunodetection was used as a loading control. Representative of 5 independent experiments. ( h ) β-catenin protein expression was assessed in the MCF10A cells transfected with siCTL or siβ4 (30 nM) after the treatment with 10 µM MG132 for 3 h, 6 h, 12 h, or 24 h, or with the solvent DMSO. β-actin immunodetection was used as a loading control.

Article Snippet: A stable MCF10A cell line knocked-down for the expression of the SCN4B gene, coding for Na V β4, was generated using the CRISPR/Cas9 technique, as previously described [ ] by transfection with the SCN4B Double Nickase Plasmid (sc-411001, Santa Cruz, France).

Techniques: Staining, Expressing, MANN-WHITNEY, Western Blot, Solvent, Immunodetection, Control, Transfection

Journal: Cells

Article Title: The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells

doi: 10.3390/cells10071624

Figure Lengend Snippet: Na V β4 expression prevents mesenchymal transition in MCF10A epithelial mammary cells. ( a ) Expression of genes associated with either epithelial ( CDH1 ) or mesenchymal ( CDH2 , SNAI1 , SNAI2 , TWIST , ZEB1 , VIM , ACTA2 ) phenotype by RT-qPCR in MCF10A CTL and MCF10A Crβ4 cells (n = 5–9 independent experiments). Results are expressed relatively to those of the MCF10A CTL cells. “ns” stands for no statistical difference. Statistically different: *, p < 0.05; **, p < 0.01 (Mann–Whitney rank sum test). ( b ) Representative western blots showing the protein expression of E-cadherin, N-cadherin, vimentin, α-SMA in the MCF10A CTL and MCF10A Crβ4 cells. Immunodetection of HSC70 was used as a loading control (n = 4 independent experiments). ( c ) Na V β4 protein expression was assessed by western blotting in MCF10A Crβ4 cells transfected with empty pcDNA3.1 or with the SCN4B gene in the pcDNA3.1 vector. Representative of 8 independent experiments. ( d ) Expression of genes associated with either epithelial ( CTNNB1 , CDH1 ) or mesenchymal ( CDH2 , SNAI1 , SNAI2 , TWIST , ZEB1 , VIM , ACTA2 ) phenotype by RT-qPCR in the MCF10A Crβ4 cells transfected with empty pcDNA3.1 or with the SCN4B gene in the pcDNA3.1 vector (n = 8 independent experiments). Results are expressed relatively to those of cells transfected with the empty vector. Statistically different: * p < 0.05 (Mann–Whitney rank sum test), otherwise no statistical difference.

Article Snippet: A stable MCF10A cell line knocked-down for the expression of the SCN4B gene, coding for Na V β4, was generated using the CRISPR/Cas9 technique, as previously described [ ] by transfection with the SCN4B Double Nickase Plasmid (sc-411001, Santa Cruz, France).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Immunodetection, Control, Transfection, Plasmid Preparation

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 1 | SCN4B/b4 protein is expressed in normal epithelial cells of human breast tissues and is downregulated in cancer cells. (a,b) b4 protein (expression of the SCN4B gene) was analysed by immunohistochemistry on human breast tissue samples. (a) The expression of b4 protein was strong in epithelial cells of mammary acini (some examples are indicated by the black arrows), and not in non-epithelial cells of normal breast tissues. (b) In breast cancer tissue, the expression of b4 protein was strong in normal epithelial cells of mammary acini (black arrows), but significantly reduced in cancer cells (tumour area indicated by the red arrow, ‘T’). Scale bars, 50 mm.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Immunohistochemistry

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 2 | SCN4B down regulation in human breast cancer tissues associates with poor prognosis. (a–d) SCN4B/b4 protein expression was analysed by immunohistochemistry on breast tissue microarrays. Samples were stratified in ‘no staining’, ‘weak staining’ or ‘strong staining’ groups. (a) Proportion of samples showing no (white), weak (gray) or strong (black) b4 staining in normal breast, compared with mammary hyperplasia/dysplasia and cancer (mixed grades) samples. The number of samples per condition is indicated in brackets. SCN4B/b4 protein staining was stronger in normal compared with cancer samples (w2, Po0.001), and in hyperplasia/dysplasia compared with cancer samples (w2, Po0.001). (b) SCN4B/b4 staining from indicated samples. Scale bars, 50 mm. (c) Proportion of samples showing no, weak or strong SCN4B/b4 staining in cancer samples, from grade I to III, and in lymph node metastases (LNM) samples. The number of samples per condition is indicated in brackets. b4 staining was stronger in grade I cancer samples compared with more advanced cancer samples (grades II, III and LNM) (w2, Po0.001). (d) Representative pictures of b4 staining from grade I, II and III ductal carcinoma samples. Scale bars, 50 mm. (e) Expression of the SCN4B gene in non-cancer (n ¼ 29) and in invasive breast carcinoma tissues (n ¼ 145) was analysed from The Cancer Genome Atlas (TCGA). RNA level is expressed as reads per kilobase per million (RPKM). Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. SCN4B gene was significantly downregulated in cancer tissues (Mann–Whitney rank sum test, MW, Po0.001). (f,g) Prognostic analyses of gene expression in breast cancers, performed using the Breast Cancer Gene-Expression Miner. (f) Kaplan–Meier Any Event (AE)-free survival analyses, performed on data pooled from cohorts for the expression of SCN4B gene (n ¼ 1,024 patients). AE is defined as being metastatic relapse (MR) or patient death. A weak expression of SCN4B gene (r median of the pooled cohorts) was associated with a decrease in the AE-free survival (P ¼ 0.0005). (g) Kaplan–Meier MR-free survival analyses were performed for the expression of SCN4B (n ¼ 661 patients). A weak expression of SCN4B (r median of the pooled cohorts) was associated with a decrease in the MR-free survival (P ¼ 0.0013). Cox results are displayed on the graph.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Immunohistochemistry, Staining, MANN-WHITNEY, Gene Expression

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 3 | Expression of the SCN4B/b4gene in human breast cancer cell lines and contribution to cancer cell invasiveness. (a) The expression of SCN4B gene was studied by RT–qPCR in human mammary epithelial non-cancer MCF-10A and cancer MCF7, MDA-MB-468, MDA-MB-435s and MDA-MB-231 cell lines. Results are expressed as relative to that of HPRT-1 gene (n ¼ 7–12) and presented as mean values±s.e.m. *, significantly different from MCF-10A at Po0.05 (MW). (b) The expression of SCN4B/b4 protein was studied by densitometric analysis of western blot experiments in same cells as in a. Results are given as the amount of SCN4B/b4 protein relative to that of HSC70 (n ¼ 5) and presented as mean values±s.e.m. *, significantly different from MCF-10A at Po0.05 (MW). The image on top shows a representative western blotting experiment. (c) The expression of SCNxB genes was analysed in MDA-MB-231-Luc cells by reverse transcription–PCR. Plasmids encoding human SCNxB genes were used as positive controls for PCR primers. (d) Representative western blotting experiments showing protein expression for b1 (SCN1B), b2 (SCN2B) and b4 (SCN4B) in MDA-MB-231-Luc cells. (e) Cells were transfected with scrambled siRNA (siCTL) or with siRNA directed against the expression of the SCN1B gene (siSCN1B), the SCN2B gene (siSCN2B) or the SCN4B gene (siSCN4B). The efficacy of siRNA transfection was assessed by western blotting experiments 48 h after transfection. HSC70 was used as a control for sample loading. (f) Representative images of fixed and haematoxylin-stained MDA-MB-231-Luc cells on invasion inserts. Cancer cells were transfected with scrambled siCTL or with specific siRNA. Scale bars, 50 mm. (g) Summary of cancer cell invasiveness results (n ¼ 8) for MDA-MB-231-Luc cells transfected with siCTL or siSCNxB. Results were expressed relative to siCTL and presented as mean values±s.e.m. ***, statistically different from siCTL at Po0.001 (Student’s t-test). (h) Representative image of a zebrafish embryo injected in the yolk sac with MDA-MB-231-Luc cells stained with CM-Dil and showing sites of colonization. Scale bars, 500 mm. Below is a magnification of the highlighted region containing human cancer cells (see arrows) colonizing organs of the embryo. (i) Zebrafish colonization index of siCTL or siSCN4B cells. Numbers in brackets indicate the number of embryos examined for each condition, from three different experiments. Results are presented as mean values±s.e.m. **, statistically different from siCTL at Po0.01 (Student’s t-test).

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Transfection, Control, Staining, Injection

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 5 | Loss of SCN4B/b4 expression maintains NaV1.5-mediated persistent current and dependent extracellular matrix degradation. (a) Sodium current (INa)–voltage relationships in shCTL (black squares, n ¼ 18) and in shSCN4B (red circles, n ¼ 22) cells. There was a significant difference at Po0.001 between the two conditions in the voltage range between 40 and þ 40 mV. (b) Activation (filled circles)– and availability (filled squares )– voltage relationships in shCTL (black symbols) and shSCN4B (red symbols) cells. (c) INa peak and INa persistent currents obtained from shCTL (black trace) and shSCN4B (red trace) cells for a membrane depolarization from 100 to 30 mV. (d) Mean values±s.e.m. of INa persistent currents obtained for a membrane depolarization from 100 to 30 mV from 18 shCTL and 21 shSCN4B cells. NS, not statistically different. (e) Mean values±s.e.m. of INa persistent/INa peak currents ratios obtained from 18 shCTL and 21 shSCN4B cells. **, statistically different from shCTL at Po0.01. (f) Dose–response effect of TTX on the inhibition of INa peak elicited by a membrane depolarization from 100 to 5 mV in shCTL (black squares, n ¼ 8–12) and in shSCN4B (red circles, n ¼ 7–12) cells. Data were fitted with the Hill equation and IC50 values were 2.02±0.10 and 2.24±0.11 mM for shCTL and shSCN4B cells, respectively. (g) Intracellular pH measurements using the BCECF-AM probe, in NH4Cl-acidified shCTL (black trace) and shSCN4B (red trace) cells in the absence of NaCl. NaCl (130 mM) was added at the time indicated (arrow). (h) H þ efflux measurements after the addition of NaCl in conditions similar to g (n ¼ 20). Results are expressed as mean values±s.e.m. NS, no statistical difference. (i) MDA-MB-231 shCTL or shSCN4B cells were cultured on a Matrigel matrix containing DQ-Gelatin. A ‘Matrix-Focalized-degradation index’ was calculated as being F-actin foci (red labelling, phalloidin-Alexa594) co-localized with focused proteolytic activities (green) (n ¼ 442 cells for shCTL and 448 cells and shSCN4B). Results are expressed as mean values±s.e.m. NS, no statistical difference. (j) Representative pictures showing matrix degradation areas (green spots) and F-actin foci (red spots). Merging points (coloc), which appear as white pixels, were counted. Numbers of white pixels per cell were normalized to the mean value obtained in shCTL cells. Statistics were performed using Student’s t-test.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Activation Assay, Membrane, Inhibition, Cell Culture

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 6 | The loss of SCN4B/b4 expression promotes human cancer cell migration and invasiveness in two and three dimensions. (a) Cancer cell invasiveness was assessed using Matrigel-invasion chambers from shCTL or shSCN4B MDA-MB-231 cells, in the absence ( ) or presence of the protease inhibitors GM6001 (10 mM), leupeptin (200 mM) or E64 (100 mM). Results from three to seven independent experiments are presented and were expressed relative to shCTL cells in the absence of inhibitors. Results are expressed as mean values±s.e.m. *** denotes a statistical difference from the shCTL at Po0.001, and # indicates a statistical difference from shSCN4B at Po0.05 (ANOVA). (b) Cancer cell migration of shCTL and shSCN4B cells measured by time-lapse microscopy to track the movement of cells over 180 min, 1 frame per min (n ¼ 20 representative cells in each condition). Distances are indicated in mm. (c) The speed of migration (in mm min 1) was analysed in shCTL and shSCN4B cells from time-lapse experiments and results shown were obtained from 106 and 96 cells, respectively. *** denotes a statistical difference from the shCTL at Po0.001 (MW). (d) The track length of cell migration (in mm) was analysed over 180 min in shCTL and shSCN4B cells from time-lapse experiments and results shown were obtained from 106 and 96 cells, respectively. *** denotes a statistical difference from the shCTL at Po0.001 (MW). (e) Three-dimension (3D) invasiveness of shCTL and shSCN4B cells, embedded inside Matrigel, was measured by time-lapse microscopy to track the movement of cells over 48 h (1 frame per 30 min) in the absence (CTL) or presence of the MMP inhibitor GM6001 (10 mM) (n ¼ 13 representative cells in each condition). Distances are indicated in mm. (f) The track length of 3D cell invasiveness (in mm) was analysed over 48 h in shCTL and shSCN4B cells from time-lapse experiments and results shown were obtained from 30 cells in each condition. ** and *** denote statistical difference from the shCTL, CTL condition at Po0.01 and Po0.001, respectively. ## denotes a statistical difference from the shSCN4B, CTL condition at P ¼ 0.002. y denotes a statistical difference from the shCTL, GM6001 condition at P ¼ 0.038 (Dunn’s test). (c,d and f) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Migration, Time-lapse Microscopy

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 7 | The loss of SCN4B/b4 expression promotes RhoA-dependent amoeboid cell transition and migration. (a) F-actin was stained with phalloidin-AlexaFluor594 in shCTL and shSCN4B cells and a cell circularity index was calculated (n ¼ 88 cells per condition). Results are expressed as mean values±s.e.m. ***Po0.001 from shCTL (Student’s t-test). (b) Representative SEM micrographs of shCTL and shSCN4B cells. Scale bars, 10 mm. (c) Number of filopodia-like structures per cell, counted from SEM pictures in shCTL and shSCN4B cells (n ¼ 60 and 66 cells, respectively). ***Po0.001 from shCTL (MW). (d) Number of blebs per cell, counted from SEM micrographs in shCTL and shSCN4B cells (n ¼ 82 cells per condition). ***Po0.001 from shCTL (MW). (e) Representative confocal micrographs of shCTL and shSCN4B cells for which F-actin was stained with phalloidin-AlexaFluor488 (green) and nuclei with DAPI (blue), scale bar 20 mm. For enlargements images, scale bars are 5 mm. (f) Number of filopodia per cell, counted from confocal micrographs in shCTL and shSCN4B cells (n ¼ 46 and 66 cells, respectively). ***Po0.001 from shCTL (MW). (g) Length of filopodia, measured from confocal micrographs, in shCTL and shSCN4B cells (n ¼ 1,070 and 593 filopodia, respectively). ***, statistically different from shCTL at Po0.001 (MW). (h) SEM observations of shSCN4B cell invasion 24 h after cells were seeded on a layer of Matrigel (4 mg ml 1). The coloured structure is the tip of the cell still observable above the Matrigel layer, while penetrating inside the matrix. Scale bar, 10 mm. (i) Western blots showing total and active GTP-bound forms of RhoA, Rac1 and Cdc42, pulled down by GST-RBD in shCTL and shSCN4B cells. (j) Quantification of GTP-bound RhoGTPases (active), normalized to total protein level, and expressed relatively to that of shCTL (n ¼ 5). **, statistically different from shCTL at Po0.01 and * at Po0.05 (MW). (k) shCTL or shSCN4B cancer cell invasiveness, in the absence ( ) or presence of blebbistatin (50 mM) (n ¼ 3). Results are expressed as mean values±s.e.m. ***Po0.001 from shCTL, and ###Po0.001 from shSCN4B (ANOVA). (l) Left panel, in situ proximity ligation assays showing a strong proximity between SCN4B/b4proteins and RhoA in shCTL cells (red dots, left panel) and the absence of any proximity signal in shSCN4B cells (right panel). Scale bars, 50 mm. (c,d,f,g,j) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Migration, Staining, Western Blot, In Situ, Ligation

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 8 | SCN4B/b4 protein overexpression inhibits cancer cell invasiveness. (a) CTL, shSCN4B and oeSCN4B cancer cell invasiveness, in the absence ( ) or presence of TTX (30 mM), expressed relative to oeCTL cells in the absence of TTX (n ¼ 6). Results are expressed as mean values±s.e.m. ***, different from CTL at Po0.001, ** at Po0.01. ###, different from shSCN4B at Po0.001 (ANOVA). NS, no statistical difference. (b) Cancer cell invasiveness (n ¼ 6) from oeCTL and oeSCN4B cells, in the absence ( ) or presence of TTX (30 mM), expressed relative to oeCTL cells in the absence of TTX. Results are expressed as mean values±s.e.m. **, different from oeCTL at Po0.01. NS, no statistical difference (ANOVA). (c) INa–voltage relationships in oeCTL (black squares, n ¼ 15) and oeSCN4B (green triangles, n ¼ 43) cells. There was a significant difference at Po0.05 between the two conditions in the voltage range between 45 and þ 45 mV. (d) Activation (filled circles)– and availability (filled squares)–voltage relationships obtained in the same oeCTL (black symbols) and oeSCN4B (green symbols) cells as in c. (e) Mean values±s.e.m. of INa persistent currents obtained for a membrane depolarization from 100 to 30 mV from 15 oeCTL and 43 oeSCN4B cells. *Po0.05 from oeCTL (Student’s t-test). (f) Mean values±s.e.m. of INa persistent/INa peak current ratios in same conditions as in e. ***P ¼ 0.001 (Student’s t-test). (g) oeCTL or oeSCN4B cells were cultured on a Matrigel-composed matrix containing DQ-Gelatin, and a ‘Matrix-Focalized-degradation index’ was calculated (n ¼ 77 and 69 cells for oeCTL and oeSCN4B, respectively). ***, statistically different from oeCTL at Po0.001 (MW). (h) Cell circularity index was calculated from oeCTL and oeSCN4B cells (n ¼ 73 cells per condition). Results are expressed as mean values±s.e.m. ***, statistically different from oeCTL at Po0.001 (Student’s t-test). (i) Speed of migration (mm min 1) of oeCTL and oeSCN4B cells analysed from time-lapse experiments (n ¼ 47 per condition). ***, statistically different from oeCTL at Po0.001 (MW). (j) Western blots showing total and active GTP-bound forms of RhoA, Rac1, Cdc42, pulled down by GST- in oeCTL and oeSCN4B cells. (k) Quantification of GTP-bound RhoGTPases in oeSCN4B cells, normalized to its total protein level, and expressed relatively to that of oeCTL cells (n ¼ 4). *, statistically different from the oeCTL at Po0.05. NS, no statistical difference (MW). (g,i,k) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Over Expression, Activation Assay, Membrane, Cell Culture, Migration, Western Blot

Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 10 | SCN4B expression inversely correlates with primary tumour growth and metastatic development. (a) Top cartoon, transendothelial migration experiment. Bottom box plot, quantification of the number of shCTL, shSCN4B or oeSCN4B cancer cells migrating through the endothelium (HUVEC monolayer) and the 8-mm pore-sized filter of the migration transwell, expressed as a relative number to shCTL (4 independent experiments). (b) Top cartoon, transendothelial invasion experiment. Bottom box plot, quantification of the number of shCTL, shSCN4B or oeSCN4B cancer cells migrating through the extracellular matrix (matrigel) coating the 8-mm pore-sized filter of the invasion transwell, then endothelium (HUVEC monolayer), expressed as a relative number to shCTL (three independent experiments). (c) Bioluminescent imaging (BLI) performed in NMRI nude mice tail vein-injected with MDA-MB-231-Luc cells that do not express (shSCN4B), or which overexpress, the SCN4B protein (oeSCN4B). Representative ex vivo lung BLI, after organ isolation, at completion of the study (ninth week after cell injection). (d) BLI quantification of excised lungs from mice injected with shSCN4B cells (n ¼ 7) and mice injected with oeSCN4B cells (n ¼ 8) (MW). (e) Mean±s.e.m. in vivo BLI value of tumours (expressed in c.p.m.) as a function of time recorded in the whole body of mice. * denotes a statistical difference from the shSCN4B group at Po0.05 (Student’s t-test). (f) Representative bioluminescent images of mammary tumours in shSCN4B and oeSCN4B experimental groups (23rd week after cell implantation). (g) Immunohistochemical analyses of primary mammary tumours obtained from same mice as in Fig. 8e,f implanted with shSCN4B cells (top image) or with oeSCN4B cells (bottom image). Slides were counterstained with haematoxylin (blue labelling), and incubated with anti-mouse SCN4B/b4 antibodies and immunohistochemistry was performed using the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen (brown labelling). Scale bars, 100 mm. (h) Immunohistochemical analyses of lungs obtained from the same mice as in Fig. 6e,f, implanted with human shSCN4B cells (top image) and oeSCN4B cancer cells (bottom image). Slides were counterstained with haematoxylin (blue labelling), and human breast cancer cells were identified using anti-human cytokeratin 7 immunohistochemical (brown) labelling. Scale bars, 100 mm. (a,b,d) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Migration, Imaging, Injection, Ex Vivo, Isolation, In Vivo, Immunohistochemical staining, Incubation, Immunohistochemistry

Journal: Frontiers in Neural Circuits

Article Title: Cortical Auditory Deafferentation Induces Long-Term Plasticity in the Inferior Colliculus of Adult Rats: Microarray and qPCR Analysis

doi: 10.3389/fncir.2012.00086

Figure Lengend Snippet: Comparison of gene expression at 90 days after unilateral auditory cortex ablation: Microarray vs. RT-qPCR data .

Article Snippet: , 10909621 , Sodium channel, type IV, beta , Scn4b , NS , NS , 1.683 , NS , NS , NS , Rn01418017_m1.

Techniques: Comparison, Gene Expression, Microarray, Control, Binding Assay