Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: TM601 Time and Dose Response in U373 glioma cells . TM601 dose response of U373 glioma incubated with 0, 3, 6, and 10 μM TM601 for 1 h or 24 h and evaluated by immunocytochemistry and confocal microscopy in n = 3-4 experiments. The fluorescence intensity/cell was obtained studying 50-100 cells/each concentration of TM601 (at 1 h and 24 h) in each experiment. Presented are means and SD for each TM601 concentration.

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Incubation, Immunocytochemistry, Confocal Microscopy, Fluorescence, Concentration Assay

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Cellular localization of TM601 and Golgin-97 by immunocytochemistry . Immunocytochemistry followed by confocal microscopy showed cellular localization of TM601 (red) in relation to Golgin-97 (green) and nucleus (blue) in human cells: U373 glioma (A, B), U87 glioma (C, D), normal umbilical vascular endothelial HUVEC (E, F), normal astrocytes NHA (G, H, I) and normal fibroblast NHDF (J, K, L) cells. The figure shows a similar Golgi localization of TM601 in gliomas (U373, U87) and HUVEC cells but in NHA and NHDF cells, TM601 is seen away from the trans-Golgi. Shown are data representative of n = 8 experiments. The scale bars on the confocal images = 10 μm, magnification 1890 × (A, C, E, H, I, K, L), 4410 × (B, D, F), 630 × (G, J).

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Immunocytochemistry, Confocal Microscopy

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Cellular localization of TM601-488 and Golgi complex-TX Red (Golgi-Tracker) by direct fluorescence . Images of live cells and cellular localization of TM601-488 (green) in relation to the Golgi-complex-Texas-Red (red) detected by direct fluorescence in NHDF normal human fibroblasts (A), U373 glioma (B), and A549 lung carcinoma (C). The figure shows similar perinuclear localization in the Golgi area of TM601-488 in glioma U373 and lung carcinoma A549, but in NHDF cells, TM601-488 is seen in the cytoplasm away from the Golgi area and outside the cell membrane. The data are representative of n = 9 experiments. Scale bars = 10 μm, magnification 1260 × (A), 630 × (B), 2000 × (C).

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Fluorescence

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Cellular colocalization of TM601-488 and Lyso-Tracker-Red DND 99 by direct fluorescence . Images of live cells and cellular colocalization of TM601-488 (green) and Lyso-Tracker-Red DND 99 (red) detected by direct fluorescence in NHDF normal human fibroblasts (A), NHA normal human astrocytes (B), and U373 human glioma (C) using the Colocalization Tool (as described in the Methods). An overlay mask (white pixels) placed on each image visualizes the colocalization of pixels above the background threshold showing the highest level of white pixels in the U373 glioma (C). The following three fluorescent color channels were used: green (TM601-488), red (Lyso-Tracker- Red), and yellow (merge of red and green fluorescence) to determine the extent of colocalization of TM601 and LysoTracker by confocal microscopy. The merge red and green fluorescence in glioma cells (F) was compared to the controls of TM601-488 (D) alone or LysoTracker Red (E) alone. The images in D, E, and F are representative of n = 7 experiments. The Colocalization Tool in A, B, C was used in one out of 7 experiments evaluating total of 14-29 cells in 3 fields. Scale bars = 10 μm, magnification 630 ×.

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Fluorescence, Confocal Microscopy

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Effect of Filipin on uptake of TM601, CholeraToxin and EGF by glioma cells . Images of live U373 glioma cells treated with 1 μg/ml CholeraToxin B-555 (A, D), or 1 μg/ml EGF-TX-Red (B, E), or immunocytochemistry of U373 glioma treated with 10 μM TM601 (C, F) in presence or absence of filipin (5 μg/ml). There was no visible effect of filipin treatment on the level or pattern of staining for TM601, but EGF and cholera toxin staining was affected. Data are representative of total n = 4 - 5 experiments. Scale bars = 10 μm, magnification 1890 ×.

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Immunocytochemistry, Staining

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Effect of chlorpromazine and amiloride on uptake of TM601, dextran and transferrin by glioma cells . Images of U373 glioma cells (treated with: dextran-TX at 1 mg/ml (A), dextran-TX at 1 mg/ml in presence of amiloride 300 μM (E), TM601-488 10 μM (B), TM601-488 10 μM in presence of amiloride 300 μM (F), transferrin-TX at 35 μg/ml (C), transferrin-TX at 35 μg/ml in presence of chlorpromazine 10 μg/ml (G), TM601 10 μM (D) and TM601 10 μM in presence of chlorpromazine 10 μg/ml (H). Direct fluorescence is shown in A, B, C, E, F, G and immunocytochemistry in D, H. The level of staining of both controls, dextran and transferrin, were decreased by amiloride or chlorpromazine, respectively. Data are representative of total n = 3 experiments. Scale bars = 10 μm, magnification 1890 ×.

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Fluorescence, Immunocytochemistry, Staining

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Cellular colocalization of TM601 and clathrin light chain in chlorpromazine treated U373 glioma cells . Immunocytochemistry of U373 glioma cells followed by confocal microscopy showed cellular localization of TM601 (red) and clathrin (green) in relation to the nucleus (blue) in TM601 treated control (A) and in TM601 and 10 μg/ml chlorpromazine treated cells (B, C). The chlorpromazine treatment resulted in accumulation of TM601 in the perinuclear region (B, C). The schematic illustration of chlorpromazine action on TM601 uptake is shown (E). The cellular colocalization of TM601 (red) and clathrin light chain (green) was detected by immunofluorescence using the Colocalization Tool (D and F) as described in the Methods. An overlay mask (white pixels) placed on each image visualizes the colocalization of pixels above the background threshold (D and F). It shows that more of white pixels were observed in TM601 and 10 μg/ml chlorpromazine treated (F) than in TM601 (D) only treated glioma cells. This is due to a higher level of TM601 in the presence of chlorpromazine resulting in higher red immunofluorescence which merged with green immunofluorescence derived from clathrin. The following three fluorescent color channels were used: red (TM601), green (clathrin light chain), and yellow (merge of red and green fluorescence) to determine the extent of colocalization of TM601 and clathrin light chain by confocal microscopy. The merged red and green fluorescence in glioma cells (C) was compared to the controls of TM601 (red insert in C) alone or clathrin light chain (green insert in C) alone. Data are representative of total n = 3 experiments. The Colocalization Tool in D and F was used in one out of 3 experiments evaluating total of 15-26 cells in 3 fields. Scale bars = 10 μm, magnification 3780 ×.

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Immunocytochemistry, Confocal Microscopy, Immunofluorescence, Derivative Assay, Fluorescence

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Effect of chlorpromazine or amiloride on TM601 uptake by U373 glioma cells . Western blots of TM601 (10 μM) - treated U373 glioma cells in presence or absence of 300 μM amiloride or 10 μg/ml chlorpromazine (A). Lysates of trypsinized cells were used at 20-25 μg protein. Beta-actin was used as a sample loading control (representative data of n = 3). Chlorpromazine (Chlorp) affected the uptake of TM601 (TM) (A, B) while amiloride had no effect on TM601 level in glioma cells (A). The statistically significant 5 fold (p < 0.005) effect of chlorpromazine is shown on the graph (B). The data represent n = 3 experiments using duplicate samples/per experiment (total of 6 samples).

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Western Blot

Journal: Cancer Cell International

Article Title: Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

doi: 10.1186/1475-2867-11-27

Figure Lengend Snippet: Effect of chlorpromazine or amiloride on TM601 uptake by NHDF fibroblasts . Western blots of TM601 (10 μM) - treated NHDF fibroblasts in presence or absence of 300 μM amiloride or 10 μg/ml chlorpromazine (A). Lysates of trypsinized cells were used at 9-12 μg protein. Beta-actin was used as a sample loading control (representative data of n = 3). The statistically significant 2 fold (p < 0.05) effect of amiloride (Amilor) on TM601 (TM) level is shown on the graph (B). The chlorpromazine had a small, statistically not significant effect on TM601 level (A). The data represent n = 3 experiments using duplicate samples/per experiment (total of 6 samples).

Article Snippet: Next, the blots were immunoblotted in TBST blocking buffer containing either affinity-purified anti-TM601 at 2.4 μg/ml or mouse monoclonal anti-beta-actin antibodies at 0.4 μg/ml (Sigma-Aldrich, St. Louis MO, as a loading control, and incubated overnight at 4°C.

Techniques: Western Blot

Journal: Advanced healthcare materials

Article Title: Nanoparticle-based therapeutics for brain injury

doi: 10.1002/adhm.201700668

Figure Lengend Snippet: Summary of various nanoparticles for passive delivery for ischemic stroke and brain trauma applications

Article Snippet: [ 189 ] PLGA NPs chlorotoxin, Lexiscan, NEP1 150 nm −22 to −25 mV MCAo Mice i.v Reduction in infarct volume, improved survival and neurological outcomes (behavior test).

Techniques: Animal Model, Injection, Activity Assay

Journal: Advanced healthcare materials

Article Title: Nanoparticle-based therapeutics for brain injury

doi: 10.1002/adhm.201700668

Figure Lengend Snippet: Summary of various nanoparticle for active delivery for ischemic stroke and brain trauma applications

Article Snippet: [ 189 ] PLGA NPs chlorotoxin, Lexiscan, NEP1 150 nm −22 to −25 mV MCAo Mice i.v Reduction in infarct volume, improved survival and neurological outcomes (behavior test).

Techniques: Animal Model, Injection, Binding Assay, Imaging, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Chlorotoxin Conjugated with Saporin Reduces Viability of ML-1 Thyroid Cancer Cells In Vitro

doi: 10.1101/2019.12.20.885483

Figure Lengend Snippet: XTT assay results for unconjugated Chlorotoxin. Representation of cell viability with differing amounts of unconjugated Chlorotoxin ranging from 0 nM (NTC) to 600 nM. Error bars represent the standard deviation of each sample. There was no significant effect on ML-1 cell viability.

Article Snippet: Unconjugated Saporin and Chlorotoxin were acquired through Sigma-Aldrich.

Techniques: XTT Assay, Standard Deviation

Journal: bioRxiv

Article Title: Chlorotoxin Conjugated with Saporin Reduces Viability of ML-1 Thyroid Cancer Cells In Vitro

doi: 10.1101/2019.12.20.885483

Figure Lengend Snippet: XTT assay results for Chlorotoxin conjugated with Saporin on ML-1 cell proliferation. Dose-dependent inhibition of ML-1 thyroid cancer cell proliferation with the CTX-SAP conjugate treatment at varying concentrations up to 200 nM.

Article Snippet: Unconjugated Saporin and Chlorotoxin were acquired through Sigma-Aldrich.

Techniques: XTT Assay, Inhibition

Journal: bioRxiv

Article Title: Chlorotoxin Conjugated with Saporin Reduces Viability of ML-1 Thyroid Cancer Cells In Vitro

doi: 10.1101/2019.12.20.885483

Figure Lengend Snippet: Viability of ML-1 cells was decreased by 49.77% with treatment of 600 nM of Chlorotoxin Saporin conjugate.

Article Snippet: Unconjugated Saporin and Chlorotoxin were acquired through Sigma-Aldrich.

Techniques: