Fig. 1

Identification of miPEP133. a Schematic graph of the open reading frame (ORF) that encodes miPEP133. The ORF (labeled in red) was identified in the gene of miR34a, MIR34AHG. b Coomassie blue staining of SDS-PAGE gel that analyzed cell lysate of HEK293 cells transfected with the control plasmid (−) or the plasmid containing miPEP133 ORF (+). c Mass spectrometry analysis of the 15KDa protein band excised from SDS-PAGE gel, which confirmed the presence of 4 peptide fragments (#1–4) that match the sequence of miPEP133. d Western blot of miPEP133 in HEK293 cells transfected with the control plasmid (−) or the plasmid containing miPEP133 ORF (+). β-actin was used as loading control. e The overexpression of miPEP133 in HEK293 cells was assessed at mRNA level by RT-QPCR. Student’s t-test, *p < 0.001. f Western blot images of miPEP133 in HEK293 cells transfected with control and 3 siRNAs. siRNAs #1 and #3 knocked down the expression of miPEP133 protein. Student’s t-test, *p < 0.001. g RT-QPCR data confirmed the knockdown of miPEP133 mRNA by siRNAs #1 and #3. Student’s t-test, *p < 0.5. h Sequencing result demonstrates the CRISPR/Gas9-mediated partial deletion of miPEP133 ORF. Two clones contain the desired deletions of the ORF, MUT1 (28 bp) and MUT2 (5 bp). Red dotted lines indicate the deleted fragments. i Western blot images of miPEP133 in HEK293 cells. MUT1 and MUT2 clones both lost the expression of miPEP133 protein comparing to the parent cell line (blank) and the cells transfected with empty vectors (CN). j RT-QPCR of miPEP133 mRNA in the nuclear and cytoplasmic fractions of HEK293 cells. U6 and GAPDH were used as control genes. k Western blot of miPEP133 protein in normal human tissues