4 research outputs found
Blockade of Stx1 function reduces GBM cell proliferation <i>in vitro</i>.
<p><b>A.</b> Expression of exocytic SNARE machinery in several established GBM cell lines determined by immunoblot. <b>B.</b> Quantification of BrdU-positive nuclei in GBM cells. The transient expression of a GFP fused Stx1 dominant negative form (Stx1-DN) reduces the rate of BrdU incorporation in GBM cells (** p ≤ 0.01). Figure shows representative results of three independent experiments. <b>C.</b> U373 GBM cells with a reduced expression of Stx1 were established by ShRNA lentiviral transduction strategies. Immunoblot showing Stx1 downregulation in U373 cell populations stably expressing two Stx1a ShRNA (Sh01 and Sh02) sequences. Stx2 and Stx3 expression levels are not modified in U373 cells with their Stx1 function blocked (U373 Stx1a-Sh01/Sh02 and Stx1-DN). <b>D.</b> Quantification of BrdU incorporation in U373 GBM cells. Cells with their Stx1 function stably blocked showed a minor rate of BrdU incorporation (* p ≤ 0.05; ** p ≤ 0.01). Figure shows representative results of three independent experiments. <b>E.</b> Cell cycle profile of unsynchronized U373 GBM cells obtained by flow cytometry. The stable blockade of Stx1 function results in an increment of cell populations at G2/M. <b>F.</b> Quantification of the frequency of binucleated U373 GBM cells. Cells with their Stx1 function stably blocked show a major proportion of binucleated cells than their respective controls. <b>G.</b> Boyden chamber invasion assay of the indicated U373 cells, showing that loss-of-function of Stx1 reduces U373 cell invasion capacity (** p ≤ 0.01; *** p ≤ 0.001). Figure shows representative results of three independent experiments.</p
Blockade of Stx1 function reduces GBM cell proliferation <i>in vitro</i>.
<p><b>A.</b> Expression of exocytic SNARE machinery in several established GBM cell lines determined by immunoblot. <b>B.</b> Quantification of BrdU-positive nuclei in GBM cells. The transient expression of a GFP fused Stx1 dominant negative form (Stx1-DN) reduces the rate of BrdU incorporation in GBM cells (** p ≤ 0.01). Figure shows representative results of three independent experiments. <b>C.</b> U373 GBM cells with a reduced expression of Stx1 were established by ShRNA lentiviral transduction strategies. Immunoblot showing Stx1 downregulation in U373 cell populations stably expressing two Stx1a ShRNA (Sh01 and Sh02) sequences. Stx2 and Stx3 expression levels are not modified in U373 cells with their Stx1 function blocked (U373 Stx1a-Sh01/Sh02 and Stx1-DN). <b>D.</b> Quantification of BrdU incorporation in U373 GBM cells. Cells with their Stx1 function stably blocked showed a minor rate of BrdU incorporation (* p ≤ 0.05; ** p ≤ 0.01). Figure shows representative results of three independent experiments. <b>E.</b> Cell cycle profile of unsynchronized U373 GBM cells obtained by flow cytometry. The stable blockade of Stx1 function results in an increment of cell populations at G2/M. <b>F.</b> Quantification of the frequency of binucleated U373 GBM cells. Cells with their Stx1 function stably blocked show a major proportion of binucleated cells than their respective controls. <b>G.</b> Boyden chamber invasion assay of the indicated U373 cells, showing that loss-of-function of Stx1 reduces U373 cell invasion capacity (** p ≤ 0.01; *** p ≤ 0.001). Figure shows representative results of three independent experiments.</p
Botulinum toxin C1 impairs glioblastoma progression in vivo.
<p><b>A, B.</b> U373 cells were inoculated into the brain of nude mice in the absence or in the presence of botulinum toxin C1 (BoNT/C1), which specifically cleaves the SNARE proteins Stx1a/b and SNAP25. <b>(A)</b> Growth curve of GBM tumors determined by luciferase quantification (* p ≤ 0.05 at 40 DPI), and <b>(B)</b> scatter plot showing the individual size of the U373 GBM tumors formed after 40 DPI, demonstrating a marked reduction of GBM after BoNT/C1 treatment.</p
Disruption of the Stx1 function impairs GBM tumor progression <i>in vivo</i>.
<p>An equal number of the indicated U373 cell populations were stereotactically inoculated into the brain of athymic nude mice. The size of the tumors was estimated at different days post-inoculation by the quantification of luciferase activity in the tumor cells. <b>A, D.</b> Representative images of the luciferase signal from mice inoculated with the indicated U373 GBM cells after 40 DPI. <b>B, E.</b> Growth curves of the indicated U373 GBM tumors showing a marked reduction of the tumor sizes after impairment of Stx1 function (* p ≤ 0.05 at 40 DPI). <b>C, F.</b> Scatter plots showing the individual size of the indicated U373 GBM tumors after 40 DPI. <b>G.</b> Representative entire brain NMR images of mice inoculated with the indicated U373 cell populations after 30 DPI (arrowheads indicate the tumors formed) showing a marked reduction in the Stx1a-Sh01 and Stx1-DN cells. <b>H.</b> Scatter plot showing the area of the indicated U373 GBM tumors formed after 30 DPI. <b>I.</b> Representative confocal images from histological sections of 30 DPI brain tumors stained with anti-GFP and anti-BrdU antibodies. <b>J.</b> Quantification of BrdU-positive nuclei in 30 DPI U373 cell tumors showing that Stx1 loss-of-function reduces proliferation (* p ≤ 0.05; *** p ≤ 0.001).</p