Journal: Journal of neuro-oncology

Article Title: Brevican Knockdown Reduces Late-Stage Glioma Tumor Aggressiveness

doi: 10.1007/s11060-014-1541-z

Figure Lengend Snippet: A, Western blot analysis demonstrated that shRNA constructs designed against B/b (shB/b) effectively reduced the expression of full-length forms and proteolytic cleavage fragments in lysate and conditioned media samples relative to untreated and empty vector (pLL3.7) controls in 0627 GICs. α-tubulin was used as a loading control for lysate samples. B, A MTT assay was conducted to examine the in vitro proliferation profile of GICs expressing either control (pLL3.7) or two independent B/b knockdown lines (shB/b 1 and shB/b 2) over the course of 7 DIV. No significant difference in cell viability was detected between control and shB/b GICs for the duration of the assay. C, Sphere formation was examined at 5DIV between passages to evaluate the self-renewal and adhesive characteristics of control and B/b knockdown GICs. No differences were observed in their abilities to form spheres between passages, scale (100μM). D, E, Control and shB/b expressing GICs were plated on laminin coated glass coverslips in undifferentiated conditions for 3 DIV or differentiated conditions for 12 DIV then analyzed by immunohistochemistry. D, Nestin immunoreactivity (red) was detected at similar levels in control and B/b knockdown GICs in undifferentiated conditions, while serum differentiation caused similar decreases in nestin immunoreactivity (red), scale (50μM). Nuclei are counterstained with Hoechst (blue). E, Negligible GFAP immunoreactivity (red) and high levels of Olig2 immunoreactivity (violet) were detected at similar levels in control and B/b knockdown GICs in undifferentiated conditions, while differentiation caused similar increases and decreases in GFAP and Olig2 immunoreactivity, respectively, scale (50μM). Nuclei are counterstained with Hoechst (blue) C, D, E, in all cases, GFP expression was driven by either control or shB/b expressing plasmids.

Article Snippet: GICs were cultured in a modified Sato’s Medium [ 26 ] supplemented with 10ng/mL EGF and bFGF. shRNA-mediated knockdown of B/b siRNAs were transformed into shRNAs and cloned into the pRNAT-U6.1/Hygro vector (GenScript, Piscataway, NJ) for rodent models and the lentiviral delivery vector pLentiLox3.7 (pLL3.7) [ 27 ] (Addgene, Cambridge, MA, USA) for human GICs.

Techniques: Western Blot, shRNA, Construct, Expressing, Plasmid Preparation, MTT Assay, In Vitro, Immunohistochemistry

Journal: Journal of neuro-oncology

Article Title: Brevican Knockdown Reduces Late-Stage Glioma Tumor Aggressiveness

doi: 10.1007/s11060-014-1541-z

Figure Lengend Snippet: Control (pLL3.7) or B/b knockdown (shB/b) GICs were injected into the striatum of nude mice and collected in pairs at first sign of neurological deterioration. Animals were anesthetized and perfused with PBS followed by 4% PB-PFA. Brains were postfixed, cryo-protected in 30% phosphate-buffered sucrose and proccessed for immunohistochemistry. GFP expression by control or shB/b expressing plasmids was used to demarcate engrafted GICs. A, Total mean tumor volumes were reconstructed using the Cavalieri method. Tumor volumes are presented relative to the paired control, wherein control tumors were defined as 100. Results show that shB/b-GIC derived tumors had significantly reduced tumor volume relative to controls. (* indicates statistical significance, p =< 0.05). B, The A/P spread of resulting tumors were determined by an estimation based on the appearance of tumor cells at the most rostral and caudal positions in serially collected sections. Values for A/P spread are presented relative to the paired control, wherein control tumors were defined as 100. Results show that shB/b-GIC derived tumors had significantly reduced A/P spread relative to controls. (* indicates statistical significance, p =< 0.05). C, D, Low mag representative image shows control GIC-derived tumors at rostral (C) and central core (D) positions, scale (1mM). Nuclei were counterstained with Hoechst (Blue). D′, D″, High mag image shows lateral invasion and diffusion of control GIC derived tumor cells into the surrounding brain tissue at the tumor/stromal border at core positions, scale (100μM). E, F, Low mag representative image shows shB/b GIC-derived tumors have reduced tumor volumes, which can be observed at both rostral (E) and central core (F) positions relative to controls. However, shB/b GIC-derived tumors were still found to form large intracranial masses, scale (1mM). Nuclei were counterstained with Hoechst (Blue). F′, F″, High mag image shows lateral invasion of shB/b GIC-derived tumor cells at the tumor/stromal border at core positions which are similar to controls, scale (100μM).

Article Snippet: GICs were cultured in a modified Sato’s Medium [ 26 ] supplemented with 10ng/mL EGF and bFGF. shRNA-mediated knockdown of B/b siRNAs were transformed into shRNAs and cloned into the pRNAT-U6.1/Hygro vector (GenScript, Piscataway, NJ) for rodent models and the lentiviral delivery vector pLentiLox3.7 (pLL3.7) [ 27 ] (Addgene, Cambridge, MA, USA) for human GICs.

Techniques: Injection, Immunohistochemistry, Expressing, Derivative Assay, Diffusion-based Assay