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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Mol Cancer Res. 2020 Mar 17;18(6):891–902. doi: 10.1158/1541-7786.MCR-19-1208

Figure 4.

Figure 4.

(A) Time course analysis of the cellular localization of fluorescently labeled RNP. A549 cells were transfected with 20 pmol of fluorescently labeled RNP complex and seeded in 4-well chambers for imaging. Representative images of each time point are shown in the figure depicting localization of the fluorescently labeled RNP complex. The brightness of the images is enhanced to better visualize the localization of the RNP complex. Scale bar represents 20 μm. (B) Graphical representation of individual and dual fluorescence from FACS compared to indel formation. The graph displays the %Total of each quadrant at each time point. Dual Label RNP (Q2) represents each %Total from quadrant 2, which contains cells with both fluorescent components. INDEL represents each percentage for total indel efficiency by TIDE analysis of an unsorted bulk population transfected with the dual labeled RNP. Total Fluor (Q1+Q2+Q4) represents the sum of quadrant 1, 2, and 4 to assess the total population of cells with fluorescence. TracrRNA ATTO 550 (Q4) represents each %Total from quadrant 3, which contains cells with ATTO 550 fluorescence only. Cas9 GFP (Q1) represents each %Total from quadrant 1, which contains cells with GFP fluorescence only. (C) Western blot analysis of Cas9 maintenance in cells. A549 cells were transfected with CRISPR/Cas9 RNP targeting exon 2 of NRF2 and harvested at the indicated time points for western blot analysis using an antibody directed against spCas9. An antibody directed against GAPDH was used as the loading control.