Fig. 3

Preliminary design and functional validation of the single-vector dCasRx translation enhancement tool. A To package into lentiviral vectors, the dCasRx-eIF4GI fusion protein and its complementary DNA sequence, sgRNA, were cloned into plasmids containing both the CMV promoter and U6 promoter. B The subcellular localization of the single-vector dCasRx translation enhancement tool with/without NLS. Immunofluorescence were performed to further determine the localization of Cherry fluorescent label (magnification ×400). C After constructing the single-vector CRISPR-dCasRx-eIF4GI tool with NLS targeting FTH1 (NLS-SV-FTH1), the mRNA expression level of FTH1 were detected using qRT–PCR analysis. D The protein expression of FTH1 were verified using western blot and the bar graph shows the relative expression level of FTH1. E After constructing the single-vector CRISPR-dCasRx-eIF4GI tool without NLS targeting FTH1 (NNLS-SV-FTH1), the mRNA expression level of FTH1 were detected using qRT–PCR analysis. F The protein expression of FTH1 were verified using western blot and the bar graph shows the relative expression level of FTH1. Data are presented as the means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, no significant difference, represents P > 0.05