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. 2022 May 18;8(20):eabn7382. doi: 10.1126/sciadv.abn7382

Fig. 4. Validation of hierarchical self-uncloaking property of RNCOs in live cell.

Fig. 4.

(A) Schematic illustration of target-specific cis-recognition and cleavage of EGFRvIII mRNA for gene silencing and trans-cleavage of RNSs for burst release of DOX. (B) Immunofluorescence (20×) of EGFP and EGFRvIII expressions in U87-EGFRvIII-EGFP cells treated with PBS (control), nRNCOs, and RNCOs, respectively. Scale bar = 100 μm. (C) Quantitative analysis of EGFRvIII expression from (B). (D) FCM and (E) qRT-PCR analysis of EGFRvIII expression after the same treatment with (B). (F) Quantitative analysis of DOX release from (G) and (H). (G and H) CLSM of DOX release profiles from RNCOs-D in U87 and U87-EGFRvIII cells. Scale bar = 25 μm. Pearson correlation coefficients are shown in the co-localization scatterplots, indicating the co-localization of Cas13a with DOX. Data are means ± SD (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (***P < 0.001).