Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 1. Analysis of COL16A1 knockdown efficiency. (A) The knockdown was translated to the protein level as shown for the full-length collagen XVI chain in cell lysates in comparison to the scr control and untreated U87MG cells. E-actin. protein expression is unaffected which indicates a specific knockdown of collagen XVI. N = 4 (B) Collagen XVI inhibited cells secreted less collagen XVI into the supernatant in comparison to scr controls. Ponceau Red stained blot membrane served as loading control. N=3 (C) Knockdown efficiency of COL16A1 was analyzed with qRT-PCR and shows an average of 60% lower COL16A1 gene expression in transfected U87MG cells (si1 and si2) compared to control cells transfected with scrambled RNA (scr). N = 19, si1= siRNA 1, si2 = siRNA 2, scr = scrambled RNA = controls.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Knockdown, Comparison, Control, Expressing, Staining, Membrane, Quantitative RT-PCR, Gene Expression, Transfection

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 2. Cell morphology and number of focal adhesion contacts. (A) U87MG cells were stained using vinculin antibodies to visualize cell morphology and focal adhesion contacts. Representative images (magnification 200x and 400x) demonstrate that in collagen XVI-knockdown cells FACs were found more concentrated in the cellular periphery while in control cells FACs (white arrows) appeared to be more evenly distributed. N = 4. (B) The number of FACs per cell is significantly decreased in collagen XVI knockdown cells. The evaluation of the number of focal adhesion contacts yielded a significant decrease of 40-45% of FACs per cell after inhibition of collagen XVI. A similar reduction of focal adhesion contacts was observed for both siRNAs. N = 4; * = p < 0.05.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Staining, Knockdown, Control, Inhibition

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 3. Invasive potential of collagen XVI inhibited cells. (A) Downregulation of collagen XVI expression resulted in a reduced invasion of U87MG cells in a Boyden Chamber assay compared to scr-control cells. The number of cells migrated to the lower compartment after 4 hours was significantly decreased in knockdown cells compared to the control. N = 4; * = p< 0.05; ** = p<0.01. (B, C) Adhesion capacity after 4 hours and 6 hours was not influenced by reduced collagen XVI expression. N = 6.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Expressing, Boyden Chamber Assay, Control, Knockdown

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 4. Downstream gene expression analysis. (A) Gene expression of PCDH18, kindlin-1 and kindlin-2 was determined by qRT-PCR and evaluated using the ''Ct method with GAPDH as endogenous loading control set to 0 and U87MG cells transfected with scrambled RNA as calibrator. PCDH18 gene expression was profoundly upregulated whereas gene expression of kindlin-1 and kindlin-2 was clearly downregulated. N = 6. ***= p< 0.001. Protein expres- sion of PCDH 18 (B), Kindlin-1 (C) and Kindlin-2 (D) was determined with western blotting analysis of cell lysates. E-Actin was used as internal loading control. N=3. Control: cell lysate from mouse lung.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Gene Expression, Quantitative RT-PCR, Control, Transfection, Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 6. Flow cytometric analysis of E1-integrin expression and activation status. FACS analysis was performed to determine E1-integrin expression and activation on collagen XVI-knock- down U87MG cells and scr controls 96 hours post transfection. (A) The antibody integrin beta1D (MAB2259Z) recognizes activated E1-integrin only. Compared to controls E1-integrin was about 50% less activated on collagen XVI-knockdown cells. (B) The antibody integrin beta1 (MAB1900) binds to to- tal E1-integrins (both activated and inactivated forms). There- fore, the result demonstrates that total expression of E1-integrin was not altered on collagen XVI-knockdown cells. black line: Isotype control which is the secondary antibody without first antibody. si1= siRNA 1, si2 = siRNA 2, scr = scrambled RNA.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Expressing, Activation Assay, Knockdown, Transfection, Control