Fig. 6

Fig. 6

Graphs showing qRT-PCR analysis of aCYSLTR2, b15-PGDH, and cGLI1 mRNA expression in HCT-116 cells with stable transfection of doxycycline-regulated shCYSLTR2 cultured with or without doxycycline (1 µM) treatment followed by LTC₄ (40 nM) stimulation for 48 h. d Western blot analysis of CysLT₂R, 15-PGDH, GLI1, and SI expression in whole-cell lysates. α-Tubulin served as the loading control. e qRT-PCR analysis showing mRNA expression of GLI1 and f western blot analysis of GLI1 protein expression in HT-29 cells stimulated with or without LTC₄ and with or without AP100984 (a CysLT₂R antagonist). HPRT1 was used as the housekeeping gene, and α-tubulin was used as the loading control in the western blot assay. g Schematic illustration of colonosphere formation. GLI1 regulates the effect of LTC₄ on stemness in multicellular colonospheres. The cells were cultured in ultra-low-attachment conditions on matrigel containing serum-free medium for 14 days. h Representative images of colonospheres from HT-29 cells transfected with shCTRL, shHPGD, or shGLI1 and stimulated or not stimulated with LTC₄. Bar graphs showing the number of colonospheres formed per well and the size of colonospheres with or without LTC₄ stimulation and comparing the shCTRL-, shHPGD-, and shGLI1-transfected groups. qRT-PCR analysis of the stemness markers iDCLK1, jGLI1, and mLGR5 in colonospheres derived from shCTRL-, shHPGD-, or shGLI1-transfected HT-29 cells with or without LTC₄ stimulation for 48 h. k Western blot analysis showing the expression of DCLK1, 15-PGDH, and GLI1 in transfected HT-29 cells as indicated. α-Tubulin served as the loading control. qRT-PCR analysis of lDCLK1 and nLGR5 in matched pairs of mucosa (M) and tumour (T) tissues from CRC patients (n = 17). HPRT1 was used as the housekeeping gene for normalization of the qRT-PCR gene expression data. Data represent the mean ± SEM from 4 to 5 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.