Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Lis1 and CD133 gene expression in neurosphere-like U87 cells. (A) Exposure of U87 glioma cells to stem-conditioned medium (SM) induces phenotypic modifications resulting in neurospheres after five days, in both regular U87 cells (left) and shLis1-U87 cells (right). The expression of Lis1 (B) and CD133 (D) is induced in U87 cells exposed to SM, with the highest level at day 5 of incubation. (C) As expected, silencing Lis1 gene in U87 cells (shLis-U87) inhibits its induction in cells incubated with SM. CD133 induction is almost abrogated in shLis-U87 cells (E) compared with U87 cells (D). Lis1 is highly expressed in CD133+ cells isolated from U87 cell line and primary glioblastoma (HTC1 and HTC2) cell cultures in normal culture medium (CM) or SM (F) . Data show enrichment up to 60 fold in Lis1 expression in CD133 + fractions for cells grown in CM and up to 32 fold in cells incubated in SM. Lis1 expression in CD133+ fraction isolated from U87 cells grown in CM is 32 times higher than that of CD133- fraction (U87 columns); in CD133+ fraction isolated from U87 incubated in SM Lis1 expression is 35 times higher than in CD133 negative cells from the same culture. The negative control is represented by CD133+ cells isolated from shLis-U87 incubated in CM or SM for which Lis1 was not increased as compared with CD133- cells (shLis-U87/CM and SM columns).

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Gene Expression, Expressing, Incubation, Isolation, Negative Control

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Proliferation of irradiated U87 and shLis-U87 cells. Cells having Lis1 silenced or not were irradiated with X-ray doses from 5 to 50 Gy. Cells seeded at a density of 1x10 4 cells/well, in quadruplicates or triplicates in E-plates and placed in xCelligence RTCA instrument, were followed-up for 100 hours (A) . Alternatively, irradiated or not irradiated cells were seeded in 24-well plates and the DNA amount per well was determined using Hoechst 33342 (B) . Both methods showed that irradiated U87 cells recovered better their proliferative capacity than shLis-U87 cells.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Irradiation

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Cell adhesion, migration and proliferation of CD133 + cells isolated from U87 and shLis-U87 cells . CD133+ cells were isolated from control U87 and shLis-U87 cells. The purity of the fraction is revealed by higher CD133 expression (assessed by RT-PCR) in CD133+ fraction as compared with CD133- fraction (A) . CD133+ cells isolated from control U87 (blue circles) or from shLis-U87 (red squares) cultures were subjected to functional assays using xCELLigence Real-Time Cell Analysis instruments. The data representing the recorded cell index at different times show that CD133+ cells isolated from shLis-U87 culture present two times lower (B) adherence to the surface, (C) migratory potential and (D) proliferative rate, as compared with the those isolated from control U87 culture.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Migration, Isolation, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, Cell Analysis