Construction and characterization of recombinant adenovirus that expresses mouse LPAATβ or knocks down human LPAATβ.

<p><b>A</b>. Schematic representation of adenoviral shuttle vector that overexpresses LPAATβ driven by CMV promoter (for making AdR-LPAATβ). The shuttle vector also expresses RFP marker. <b>B</b>. Schematic representation of adenoviral pSOS shuttle vector that expresses siRNA driven by dual opposing promoters (for making AdR-siLPAATβ). The pSOS shuttle vector also expresses RFP marker. The four siRNA targeting sites against human LPAATβ are listed. <b>C</b>. Adenovirus-mediated effective transduction of human osteosarcoma cells. Optimal titer of the recombinant adenoviruses AdR-LPAATβ and AdR-siLPAATβ were used to infect 143B cells. The expression of red fluorescent protein (RFP) was examined at 48 h after infection. <b>D</b>. Characterization of AdR-LPAATβ-mediated overxpression and AdR-siLPAATβ-mediated knockdown. Subconfluent 143B cells were infected with AdR-LPAATβ, AdR-siLPAATβ, or AdRFP control for 48 h. Total RNA were collected and subjected to RT-PCR. The resultant cDNA was subjected to semi-quantitative PCR using primers specific for mouse LPAATβ mRNA (for AdR-LPAATβ infection) or human LPAATβ mRNA (for AdR-LPAATβ infection). GAPDH was used as an internal control to normalize all samples.</p

Effect of LPAATβ on osteosarcoma cell proliferation.

<p><b>A</b>. Crystal violet staining assay for cell viability. 143B cells were seeded in 12-well plates and infected with an optimal titer of AdR-LPAATβ, AdR-LPAATβ, or AdRFP control. Viable cells were subjected to crystal violet staining at 5 days after infection. Representative duplicate staining is shown. <b>B</b>. Quantitative analysis of the crystal violent staining assay. Relative staining intensities were measured by using ImageJ software. “*” <i>p-value <0.05</i>. <b>C</b>. MTT cell proliferation. 143B cells were seeded in 96-well plates and infected with an optimal titer of AdR-LPAATβ, AdR-LPAATβ, or AdRFP control (in triplicate). At the indicated time points after infection, cells were subjected to MTT assay to determine relative proliferative activity. “*” <i>p-value <0.01</i>.</p

Histological evaluation of xenograft osteosarcoma tumors.

<p>Tumors were retrieved from animals and subjected to paraffin-embedded. Sections were subjected to Hemotoxylin & Eosin staining (top panel) and PCNA antibody immunohistochemical staining (bottom panel). Representative results are shown.</p