Fig. 4

The pharmacological treatment with BODIPY-THIF or ablation of STAT1 via a CRISPR/Cas9-based strategy reduces stemness in colorectal cancer. A Correlations between STAT1 and colorectal cancer stem cell (CSC) markers in patients with colorectal adenocarcinoma using TCGA database (https://gdc.cancer.gov/). B The expansion of colonospheres was evaluated on days 1, 3, 5, 7 and 9. HCT116 cell morphology was monitored by microscopy. Scale bars: 100 μm. Single-cell suspensions of cells were cultured under bFGF ( +), EGF ( +) and serum-free conditions, allowing them to grow as colonospheres for 9 days. C The mRNA expression levels of stemness genes were measured by qPCR. mRNA expression was normalized to GAPDH expression. **p < 0.01; ***p < 0.001. D Role of STAT1 in stemness in CRC cells. STAT1 KO HCT116 cells were generated by targeting the STAT1 gene using the CRISPR/Cas9 strategy. STAT1 WT and STAT1 KO cells were cultured in an ultra-low-attachment 6-well plate for 9 days. Cell morphology was monitored by microscopy (upper panel). Scale bars: 100 μm. The number of colonospheres per well was calculated (lower panel). **p < 0.01; ***p < 0.001. E HCT116 colonospheres were dissociated into single cells and plated at a density of 5 × 10⁴ cells/well in a 6-well ultra-low-attachment plate. Cells were treated with 10 or 50 μM BODIPY-THIF for 48 h. Cell morphology was monitored by microscopy (left panel). Scale bars: 100 μm. The number of colonospheres was calculated (right panel). **p < 0.01; ***p < 0.001. F HCT116 colonospheres were dissociated into single cells and plated at a density of 5 × 10⁴ cells/well in a 6-well ultra-low-attachment plate. Cells were treated with 10 or 50 μM BODIPY-THIF for 48 h. The mRNA expression levels of stemness genes were measured by qPCR. mRNA expression was normalized to GAPDH expression. *p < 0.05; **p < 0.01; ***p < 0.001