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Fig. 5
Transient knockdown of ANKRD45 with the CRISPR/Cas9 system results in cell apoptosis. (A) Genomic structure of the human ANKRD45 gene and sequences of two sgRNAs. Red arrows indicate the position and direction of the two sgRNAs. (B) Efficiency of the two sgRNAs analyzed by T7 endonuclease I digestion (T7EI). (C) Western blotting results showing the knockdown efficiency of two ANKRD45-targeting sequences. Both targets led to the reduction of ANKRD45 expression and increased the level of the cleaved PARP. (D–F) DIC images showing Hep3B cells transfected with control or ANKRD45 targeting constructs containing different sgRNAs as indicated. Arrowheads represent the apoptotic cells. (G) Flow cytometry analysis of Annexin V-FITC and propidium iodide staining of Hep3B cells transfected with sgRNA or empty vectors. (H) Bar graph showing the percentages of Annexin V positive cells at 72 h and 96 h after transfection with control or sgRNAs as indicated. * p < 0.05, ** p < 0.01, Scale bar: 25 μm.