Effects of Growth Factors and Basement Membrane Proteins on the Phenotype of U- 373 MG Glioblastoma Cells as Determined by the Expression of Intermediate Filament Proteins  Sadath Sultana, Rixin Zhou, Madhu S. Sadagopan, Omar Skalli  The American Journal of Pathology  Volume 153, Issue 4, Pages 1157-1168 (October 1998) DOI: 10.1016/S0002-9440(10)65660-X Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 1 a: Coomassie Blue-stained SDS-polyacrylamide gel loaded with whole cell extracts of U-373 MG (lane 1), U-87 MG (lane 2), U-118 MG (lane 3), and Hs 683 (lane 4) cells. Molecular mass are indicated in kilodaltons at the left of the gel. b-d: Blots of gels similar to a incubated with the following primary antibodies: anti-nestin (b), anti-GFAP (c), and anti-vimentin (d). Note that U373-MG is the only cell line to co-express GFAP and nestin. The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 2 Double immunofluorescent staining of U-373 MG glioblastoma cells with vimentin (a) and nestin (b) antibodies. Nestin antibodies stain a filamentous network similar to that stained by anti-vimentin. Scale bar = 10 μm. The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 3 Effect of growth factors on GFAP protein levels in U-373 MG cells.a: Blot corresponding to a gel loaded with equal amounts of proteins; b: blot corresponding to a gel loaded with equal number of cells. In a and b, the lane labeled C shows the result for the control sample, and subsequent lanes show the results for cells treated with different growth factors as indicated above each lane. c: Histogram showing the changes in GFAP protein levels in growth factor treated cells relative to control cells. Values are calculated from seven different experiments and are expressed as means ± SEM. Black bars represent values determined by comparing blots obtained from gels loaded with equal amounts of proteins, and gray bars represent values determined by comparing blots obtained from gels loaded with equal number of cells. Changes that are statistically different from control are indicated by * (P ≤ 0.001) or # (P = 0.005). The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 4 Effect of growth factors on vimentin protein levels in U-373 MG cells.a: Blot corresponding to a gel loaded with equal amounts of proteins; b: blot corresponding to a gel loaded with equal number of cells. In a and b, the lane labeled C shows the result for the control sample, and subsequent lanes show the results for cells treated with different growth factors as indicated above each lane. c: Histogram showing the changes in vimentin protein levels in growth factor treated cells relative to control cells. Values are calculated from seven different experiments and are expressed as means ± SEM. Black bars represent values determined by comparing blots obtained from gels loaded with equal amounts of proteins, and gray bars represent values determined by comparing blots obtained from gels loaded with equal number of cells. Changes that are statistically different from control are indicated by * (P ≤ 0.001), # (P ≤ 0.005) or ## (P = 0.013). The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 5 Effect of growth factors on nestin protein levels in U-373 MG cells.a: Blot corresponding to a gel loaded with equal amounts of proteins; b: blot corresponding to a gel loaded with equal number of cells. In a and b, the lane labeled C shows the result for the control sample and subsequent lanes show the results for cells treated with different growth factors as indicated above each lane. c: Histogram showing the changes in nestin protein levels in growth factor treated cells relative to control cells. Values are calculated from seven different experiments and are expressed as means ± SEM. Black bars represent values determined by comparing blots obtained from gels loaded with equal amounts of proteins, and gray bars represent values determined by comparing blots obtained from gels loaded with equal number of cells. Changes that are statistically different from control are indicated by * (P = 0.022) or # (P = 0.015). The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 6 Effect of BM proteins on GFAP protein levels in U-373 MG cells.a: Blot corresponding to a gel loaded with equal amounts of proteins; b: blot corresponding to a gel loaded with equal number of cells. In a and b, the lane labeled C shows the result for the control sample, and subsequent lanes show the results for cells plated on collagen IV (COL), laminin (LMN), or fibronectin (FN). c:Histogram showing the changes in GFAP protein levels in cells plated on BM proteins relative to control cells. Values are calculated from seven different experiments and are expressed as means ± SEM. Black bars represent values determined by comparing blots obtained from gels loaded with equal amounts of proteins, and gray bars represent values determined by comparing blots obtained from gels loaded with equal number of cells. Changes that are statistically different from control are indicated by * (P ≤ 0.001). The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 7 Effect of BM proteins on vimentin protein levels in U-373 MG cells.a: Blot corresponding to a gel loaded with equal amounts of proteins; b: blot corresponding to a gel loaded with equal number of cells. In a and b, the lane labeled C shows the result for the control sample, and subsequent lanes show the results for cells plated on collagen IV (COL), laminin (LMN), or fibronectin (FN). c:Histogram showing the changes in vimentin protein levels in cells plated on BM proteins relative to control cells. Values are calculated from seven different experiments and are expressed as means ± SEM. Black bars represent values determined by comparing blots obtained from gels loaded with equal amounts of proteins, and gray bars represent values determined by comparing blots obtained from gels loaded with equal number of cells. Changes are not statistically different from control. The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 8 Effect of BM proteins on nestin protein levels in U-373 MG cells.a: Blot corresponding to a gel loaded with equal amounts of proteins, b: blot corresponding to a gel loaded with equal number of cells. In a and b, the lane labeled C shows the result for the control sample, and subsequent lanes show the results for cells plated on collagen IV (COL), laminin (LMN), or fibronectin (FN). c:Histogram showing the changes in nestin protein levels in cells plated on BM proteins relative to control cells. Values are calculated from seven different experiments and are expressed as means ± SEM. Black bars represent values determined by comparing blots obtained from gels loaded with equal amounts of proteins, and gray bars represent values determined by comparing blots obtained from gels loaded with equal number of cells. Changes that are statistically different from control are indicated by * (P ≤ 0.001) or # (0.001<P ≤ 0.005). The American Journal of Pathology 1998 153, 1157-1168DOI: (10.1016/S0002-9440(10)65660-X) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions