(A) Immunocytochemical fluorescence analysis of U251, U87, 4910 and 5310 neurospheres (passage 10). The majority of neurospheres are immunopositive for CD133, CD44, GFAP, Ki-67, Msi-1, Nestin, Sox2, Stro-1 and Tuj-1. Inset pictures show DAPI (blue) staining. Bar = 100 μm. (B) Double positive co-expression of CD133⁺ GSCs isolated from U251, U87, 4910 and 5310 neurospheres were studied by flow cytometry. CD133-sorted cells from all four GSCs were labeled individually with CD44, Stro-1, Sox2 and Msi-1 with their respective antibodies. In all the experiments, CD133 was conjugated with Alexa Fluor-594 (red), and CD44, Stro-1, Sox2 and Msi-1 was conjugated with Alexa Fluor-488 (green). The experiment was performed in duplicate, and error bars represent standard deviation. (C) Expression levels of various glioma stem cell markers and other molecules were analyzed in U251, U87, 4910 and 5310 neurospheres in comparison to their respective non-GSC cells using semi-quantitative RT-PCR. β-actin served as an internal control for equal loading of the PCR products. Results presented are representative images of three independent experiments (n=3). (D-E) Western blot analysis of various neuronal and EMT makers in U251, U87, 4910 and 5310 neurospheres. Data presented here are a representation of three individual experiments (n=3). All cell lysates were obtained from cells at passage 10.

Approximately 1×10⁵ U251 (A), U87 (B), 4910 (C), and 5310 (D) cells were transfected with either siTwist or si-Sox2. Samples were harvested after 72 hrs. siTwist1 and siSox2 expression was determined by staining with the mAb to human Twist1, Sox2 or isotype control mouse or goat IgG₁, and then followed by a secondary antibody goat anti-mouse IgG or donkey anti-goat IgG conjugated to Alexa Fluor red or green dye. The background was subtracted using different isotypic controls for respective antibodies. In overlays under Figures 4A-4D, purple color represents the negative control, green color indicates the siTwist or siSox2 treatment, orange color represents Sox2 expression, and red color indicates Twist1 expression. Bar graph (E) represents the expression level of Twist1 in siTwist1 knock down (F) represents the expression level of Sox2 in siSox2 knockdown (G) represents the expression level of Sox2 in siTwist1 knock down while (H) represents the percent expression levels of Twist1 in siSox2 knock down as quantified from the above contour plots. Cells were analyzed with a FACSCalibur flow cytometer and CellQuest software.

(A) Immunostaining analysis from neurospheres generated from GSC-enriched populations (U251, U87, 4910 and 5310 cells) shows Twist1 staining that appears to correlate with high Sox2 expression. Photographs depicted show Twist1 (red) is co-expressed/associated with Sox2 (green). All nuclei are counterstained with DAPI in blue. Scale bars represent 100 μm. (B) Immunostaining of GBM mouse xenografts (U251 and 5310) demonstrate that Twist1 (red) is expressed in the necrotic area and is co-expressed with Sox2 (green). Scale bars represent 50 μm. DAB immunohistochemical analysis of Twist1 and Sox2 expression in U251 GSC and 5310 GSC xenografts in nude mice brains. (C) Representative microphotographs of U251 and 5310 GSCs injected intracranially, which show infiltrative/migratory nature of xenograft-derived tumors. Insets in figures are representative H&E stained sections. Immunohistochemical staining was done using specific antibodies for Twist1 and Sox2 (dark brown in color). Scale bar represents 100 μm.

(A-B) Representative immunohistochemical staining of Twist1 and Sox2 in human GBM patient specimens and normal brain. Bar = 200 μm. Strong positivity of Twist1 and Sox2 was detected in five specimens (hGBMs 2,4,5,6 and 7). Negative staining was seen in normal human brain samples. (C-D) Specific expression of Twist1 and Sox2 in U251 and 5310 mouse xenografts models. Strong positivity of Twist1 and Sox2 was detected by H&E staining. Bar = 200 μm;. (E) GBM tissue specimen lysates for western blot analysis were prepared as described previously [30]. In vivo expression was studied by loading equal amounts of protein (40 μg) from tissue lysates onto 10-12% SDS-PAGE gels. (F) Semi-quantitative RT-PCR analysis was done to determine the expression of various stem cell markers in human GBM-derived biopsies. All determinations were done in duplicate.

DAB immunocytochemistry was done to study the phenotypical changes of the U251, U87, 4910 and 5310 GSC when treated with (A) Twist1 siRNA and Sox2 siRNA. Cell lystates (40 μg protein for each sample) obtained by treating U251, U87, 4910 and 5310 GSCs with (B) siTwist and (C) siSox2 were loaded onto 8-12% SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with respective antibodies showing MET transition. (D-E) Nuclear extracts of the same samples were subjected to Western blotting (n=3). GSC (+) indicates the lysate obtained from scrambled vector in the immunoblots.

(A) Co-culture experiments showing induction of MET transition by hUCBSC in GSCs. (B) Various stages of cell cycle of GSCs influenced by hUCBSC. In all of the GSCs, cell cycle was arrested at the G2-M stage. (C) Bar graph showing the results of (B). Western blot analysis showing MET pathway molecules in U251, U87, 4910 and 5310 GSCs when co-cultured with hUCBSC (D) in vitro (72 hrs) and (E) in vivo. (F) Semi-quantitative RT-PCR analysis showing MET transition of GSCs.