Fig 7. KRT17 knockout inhibited tumor growth.

(A) Confirmation of KRT17 mutation in the KRT17-knockout cells (HSC3-KO). HSC3 was transfected with pSpCas9(BB)-2A-GFP carrying the KRT17 target sequence in KRT17 exon 3 and cloned by limited dilution following fluorescence activated cell sorting of transfected cells. Six independent clones, in which KRT17 protein expression was absent, were established. Of those, one clone (HSC3-KO) exhibited homo-allelic single-base deletion as revealed by PCR and direct sequencing of the genomic DNA, resulting in a frame shift and a premature stop codon. +259 and +278 denote the nucleotide positions corresponding to KRT17 cDNA when the A of the ATG of the initiator methionine codon is designated as position +1. (B) Western blot analysis of HSC3 and HSC3-KO, demonstrating the absence of KRT17 and reduced expression of phosphorylated pAKT1, MTOR, and pEIF4EBP1 in HSC3-KO. (C) Reduced SLC2A1 expression and glucose uptake in HSC3-KO, as revealed by flow cytometry. (D) Cells (5 x 10⁵) of HSC3 or HSC-KO were subcutaneously injected into the cephalic skin of nude mice (n = 4). One mouse transplanted with HSC3 died of an unknown cause on day 10. HSC3-KO cells developed smaller tumors than HSC3. The photographs were taken on day 15. The tumor areas are encircled by yellow dashed lines. (E) The tumor area was calculated following elliptic substitution of the macroscopic tumor margin using the photograph of the vertical view. The bold lines depict mean tumor areas. The error bars represent standard errors. (F) Immunohistochemical examination of the HSC3-KO tumor developed in the nude mice, confirming negative expression of KRT17. Since the antibody recognizes both human and mouse KRT17, the physiological expression of KRT17 was observed in the hair follicles. Scale bar, 200 μm. (G) Histology of the HSC3 tumor and the HSC3-KO tumor. The HSC3 tumor was composed of medium-to-large-sized tumor nests, whereas the HSC3-KO tumor was composed of small islands. Scale bar, 200 μm. (H) Immunohistochemical expression of MKI67 in the HSC3 tumor and the HSC3-KO tumor. Note that there were fewer cancer cells in the HSC3-KO photograph than in the HSC3 photograph. Scale bar, 200 μm. (I) The Ki-67 labeling indices (LI) were calculated as the percentage of MKI67-positive nuclei in the cancer cells after counting at least 1,000 tumor cells at X200 magnification. The tumor areas that were closest to the epidermis were used for analysis.