Figure 3

siRNA reverse transfection on CSMAs. (A) siRNA mediated gene silencing on CSMA was tested with U-2OS sarcoma cells expressing a destabilized TurboGFP. Silencing efficacy of different siRNA concentrations was compared with negative control siRNA and a control siRNA for CDC2. Composite (TuGFP = green, DNA = blue) and single channel confocal laser microarray scanned images of a U-2OS array with three 25 replicate spot sub-grids of siTuGFP and siCDC2 and two control siRNA sub-grids. Scale bar 2 mm. (B) Top: Automated image analysis was used to segment nuclei of individual cells and fixed area spanning 15 pixels outside nucleus was used to measure the cytoplasmic signal for TuGFP. Middle: 20× microscopic images of control siRNA (UL) and TuGFP siRNA spots (UR = 10 ng/μl, LL = 20 ng/μl, LR = 30 ng/μl). Quantification of cytoplasmic TuGFP against nuclear DAPI intensity indicated an up to 90% transfection efficacy with 30 ng/μl siRNA. Scale bar 100 μm. (C) Top: Image based cytometry analysis of integrated nuclear DAPI intensity (nuclear area/DAPI mean intensity) was used for cell cycle analysis of siCDC2 and siTuGFP transfected cells. Coloured boxes indicate cells classified as G1-S (red), G2 (Blue) and M (purple). Inhibition of CDC2 induced a prominent G2 arrest with all siRNA concentrations in comparison to control or TuGFP. (D) Top: Microarray scanned images of a CSMA with PC-3 cells stained for ITGA5 and ITGB1 following 48 h transfection with control and ITGA5 siRNA. Top right: Based on microscopic image analysis of ITGA5 signal against DAPI counterstaining of the cells, an up to 75% knockdown was achieved across the 25 replicates with 20 ng/μl ITGA5 siRNA. Bottom: 20× microscopic images of the control and ITGA5 siRNA spots. Scale bar 2 mm and 100 μm.