Fig. 2

Effects of KYA1797K on the CSC properties of CRC cells harboring APC and KRAS mutations. DLD-KRAS-MT cells were seeded at a density of 1 × 10⁴ cells/mL in ultra-low attachment plates for spheroid culture and treated with DMSO or KYA1797K (25 μM). a Immunoblot analyses of DMSO- or KYA1797K-treated spheroids using the indicated antibodies. b Representative images of spheroids with DMSO or KYA1797K treatment on days 3 and 7 (N = 3). Scale bar represents 200 μm. c, d The numbers (c) and sizes (d) of spheroids were measured using Image J v1.47 software. e Relative mRNA levels of the CSC markers LGR5, CD44, CD133, and CD166 in spheroids treated with DMSO or KYA1797K. f ICC analysis of spheroids treated with DMSO or KYA1797K using the indicated antibodies. Scale bar represents 50 μm. g FACS analyses of CD44, CD133, and CD166 triple-positive cells in spheroids treated with DMSO or KYA1797K. h Immunofluorescent IHC analyses of tumors from DLD-KRAS-MT xenograft mice treated with vehicle or KYA1797K using the indicated antibodies. Scale bar represents 20 μm. Quantification of fluorescence signals were performed using Zen software. In (c), (d), (e), (g), and (h), results are presented as mean ± SD of three independent experiments. n.s. no significance, * P < 0.05, ** P < 0.01, *** P < 0.001