Fig. 3. Transfection of CD44 sgRNA-Cas9-GFP significantly inhibits CD44 expression. A. Fluorescence analysis show that most of KHOSR2 and U-2OSR2 cells transfected with CD44-Cas9-GFP or pEGFP-N3 plasmids contain green fluorescence, which suggests that KHOSR2 and U-2OSR2 cells were successfully, introduced CD44-Cas9-GFP or pEGFP plasmids. This assay was repeated three times. B. Western blots showing CD44-Cas9-GFP system could effectively knock out CD44. The western bolt was performed in triplicate. C. Immunofluorescence photos for CD44 (red) and nuclei (blue) of KHOSR2 and U-2OSR2 cells transfected with CD44-Cas9-GFP plasmids. This assay was repeated twice.