Journal: Cell Communication and Signaling : CCS
Article Title: Modulating voltage-gated sodium channels to enhance differentiation and sensitize glioblastoma cells to chemotherapy
doi: 10.1186/s12964-024-01819-z
Figure Lengend Snippet: Expression of SCN1A is associated with worse survival in glioblastoma patients and correlates with GSCs markers: ( A ) Pooled data for the 288-hours proliferation assays of proneural GSCs in the control condition (circles) and in the presence of TMZ 3µM (squares). Inset: the same pool of data but for the time window zoomed in the interval from 0 to 96 h. ( B ) Average I-V Plot from − 70 mV to 70 mV acquired in whole-cell patch clamp showing a significant inward current upregulation in TMZ-resistant cells (b, TMZ 3µM) compared to non-treated cells (a, Control). Inset: representative inward current traces recorded at + 10 mV for the two conditions. ( C ) Sensitivity to TTX and QX-314 revealed the nature of the recorded inward current. On top, the Na v -mediated nature of the current was assessed with bath perfusion of the specific blocker Tetrodotoxin (TTX 1µM). Representative trace in control (black) and after TTX perfusion (red). On the bottom, the Na v -mediated current was also tested with intracellular dialysis of the lidocaine derivative QX-314 500 µM, which blocks the channel from the cytosolic compartment. Representative traces in control (black) and after QX-314 dialysis (red) are also displayed. After both treatments, the inward current was significantly abolished, thus confirming the current’s identity. On the right, average pool data for all the recorded cells in control, with TTX and QX-314 perfusion. ( D ) Immunoreactivity to the Pan-Na v antibody (green channel), E-Cadherin antibody (red channel) and nuclear staining (Hoechst, blue channel) in control condition (CTR) and after 288 h TMZ 3 µM treatment. Scale bar is 50 μm. ( E ) Optical density (OD) violin plots illustrating single-cell measurements in CTR condition and after 288 h TMZ 3 µM treatment (upper panel). Pool data for the percentage of cells positive to Na v for the two conditions (lower panel).( F ) Bulk analysis of SCN1A mRNA expression levels in three different GBM subtypes. ( G ) Whole-cell electrophysiological recordings were performed on different subtypes of GSCs, and the Na v -mediated current was recorded. On the top, representative traces are recorded at different holding potentials from a proneural line. The corresponding voltage steps applied are displayed. On the bottom, the average I-V plot from − 70 mV to 70 mV for all the proneural recorded primary cell lines. (left) Recordings of adherent and suspended proneural GSCs. (right) Recordings of the classical and mesenchymal cell line. ( H ) Kaplan-Meier survival curve of GBM patients with a proneural subtype. The cut-off was set at 7.7 (log2). ( I ) SCN1A mRNA expression level positively correlates with some stemness markers. The slope value of the linear fitting and the significance of the correlation are displayed. Conversely, SCN1A negatively correlates with markers associated with proliferation and differentiation. ( J - L ) Immunohistochemical staining for different GSC markers (CD133, SOX2) and Nav in various GBM patient biopsies. Panels a and b show representative images of staining for Hoechst and the GSC marker, and Hoechst and Nav, respectively. Panel c shows the merge of the previous two panels. Scale bar is 20 μm. Panel d shows a more defined FOV taken from panel c. Scale bar is 20 μm. The white arrows indicate cells positive for both Nav and the GSC markers
Article Snippet: The following primary antibodies were used for immunostaining: mouse anti-pan-Nav sodium channel (1:250, Sigma Aldrich, Cat. No. S6936), rabbit anti-SOX2 (1:700, Cell Signaling, Cat. No. 3579), and rabbit anti-CD133 (1:200, Cell Signaling, Cat. No. 60577).
Techniques: Expressing, Control, Patch Clamp, Staining, Immunohistochemical staining, Marker
Alomone Labs is a verified supplier 
Alomone Labs manufactures this product 
