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

Journal: Experimental and Therapeutic Medicine

Article Title: Cell proliferation and invasion ability of human choriocarcinoma cells lessened due to inhibition of Sox2 expression by microRNA-145

doi: 10.3892/etm.2012.781

Figure Lengend Snippet: miR-145 and Sox2 expression in different groups. (A) The human Sox2 microRNA (miRNA) 3′-untranslated region (3′-UTR) contains miR-145 binding sites. The mature miR-145 sequences of multiple species were analyzed and contrasted using bioinformatics tools. The typical secondary structure of precursor miRNAs (pre-miRNAs) was compared with miR-145. Pre-miRNA contains stem-loop and hairpin structures and the common binding site is located in an unstable region with a multi-branching loop-like RNA structure. Mature miRNAs are bound in the 3′-UTR of the target gene. Complementarity between miR-145 and the putative human Sox2 3′-UTR site target (1–20 bp downstream) showed that the conserved bases of the putative miR-145 target sequence are present in the human Sox2 3′-UTR. (B) The expression of miR-145 and its interference with the target gene Sox2 were assessed by luciferase assays. Wild-type (wt) reporter or mutated control luciferase plasmids were transfected into NIH-3T3 cells with miR-145 or mutant miR-145 expression viruses. Luciferase activity within the Sox2 3′-UTR sites was inhibited by miR-145 ( ** P<0.01 vs. pLL3.7; #P>0.05 vs. pLL3.7; n=3). (C) Northern blot hybridized signals of miR-145 in human choriocarcinoma cells. Northern blot hybridized signals of miR-145 in (Ca) JAR cells and (Cb) JEG-3 cells. Northern blot analysis showed a weaker hybridized signal in mutant miR-145-transfected cells than in wt miR-145-transfected cells. The human U6 probe was used as a loading control. (D) Sox2 mRNA expression assay in different human choriocarcinoma cells by quantitative real-time PCR (qRT-PCR). qRT-PCR indicated lower expression of Sox2 mRNA in the (Da) wt miR-145-transfected JAR cells and (Db) wt miR-145-transfected JEG-3 cells than in the corresponding untransfected or mutant miR-145-transfected cells. Relative mRNA expression is shown after normalization to 18S rRNA, which served as an internal control. (E) Western blot showing the expression of Sox2 in various groups. Western blots showing the expression of Sox2 in (Ea) JAR cells and (Eb) JEG-3 cells. Sox2 levels were significantly higher in the untransfected or mutant miR-145-transfected cells than in the miR-145-transfected cells. Data indicate that exogenous miR-145 downregulates Sox2 expression ( ** P<0.01 vs. untransfected; # P>0.05 vs. untransfected; n=3).

Article Snippet: An RNAi pLL3.7 (LentiLox 3.7) lentiviral system was used to create lentiviral vectors (Clontech, Beijing, China).

Techniques: Expressing, Binding Assay, Sequencing, Luciferase, Transfection, Mutagenesis, Activity Assay, Northern Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot