Fig 2: Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker SSEA-1 (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a, Nanog, and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. *Compared to the expression level of cultured cESCs in passage 3, P < 0.05. (d) Nanog expression characteristic of cultured cESCs in passages 3, 5, and 20. The figure indicated the merged picture of DAPI, Nanog, and EDU staining. (e) The positive proliferation rate of cultured cESCs was maintained at a relatively high level over passages 3, 5, and 20. The bars indicated the ratio of the EDU-Nanog coexpression cell number to the Nanog expression cell number calculated by ImageXpress Micro XLS Widefield High-Content Analysis System. (f) Telomerase activity. Telomerase activities of cultured cESCs in passages 0, 3, 5, and 20 were measured in cell lysate by the TRAP assay. The cells were frozen until the TRAP was performed. HCT8 cell lysate used as a positive control and noncell lysate used as a negative control. DNA ladder (N3233 New England Biolabs) was used to indicate the size of the fragment. All results are expressed as the mean ± SD of three independent experiments. Scale bar: 100 µm.

Fig 3: Analysis of Dicer protein and mRNA in HCT116 and Dicer knockout H2-2 cells. (A) Western blot experiments to confirm gene disruption in H2-2 Dicer knockout cells compared to WT HCT116 cells. The Dicer protein is 217 kDa in humans. Two anti-Dicer antibodies (Ab14601 and Ab13502) were used to detect the Dicer protein. Tubulin was used as a loading control. (B) Amino acid sequence analysis of the mutated area from H2-2 and human Dicer. The CRISPR/Cas9 system made nine nucleotide deletions and twenty-one nucleotide substitutions (Supplementary Figure S2A). The Dicer sequence from H2-2 cells shows a different protein-peptide compared with human Dicer (NP_001182502.1: NCBI reference number). (C) Human endoribonuclease Dicer mRNA sequence (1 to 300 nt: NM_001195573.1: NCBI reference sequence). Red arrows show the forward and reverse primers used to detect Dicer mRNA by PCR. The CRISPR/Cas9 system mutated the sequence indicated with blue underline. Yellow slashes indicate the codon of amino acid. (D) RNA was extracted from HCT116 and H2-2 cells and subjected to reverse transcription either with or without (w/o RT) the RT enzyme. cDNA was amplified by PCR with a Dicer-targeted primer (red arrow in (C)). The gray arrow is the DNA band of 100 bp in size. The last lane in the gel lane loads the low molecular weight DNA ladder (New England BioLabs Inc.: catalog number N3233). Black arrow is size of 50 bp.