Fig. 3

JAK2/STAT3 signaling activation is required for cancer stemness. a HCT116 cells were cultured under attached monolayer culture and sphere-forming conditions to enrich CSCs. Immunofluorescence assays were performed to detect JAK2 expression. Blue indicates nuclei, and red indicates JAK2. Western blot analyses visualized the increase of JAK2 protein levels as well as activation of STAT3 (p-STAT3) in CSCs. b FACS analysis was performed to compare the JAK2 expression between stem marker (CD44v6, LGR5 and ALDH1A1)-positive and stem marker-negative cells. c Cells were sorted into CD44v6+ and CD44v6- fractions. Then, their intrinsic radioresistance was determined by analyzing cell viability after RT. d Cell cycle analyses were performed following radiation, and cell cycle alterations were compared between the CD44v6+ and CD44v6- fractions. e and f FACS analysis revealed (e) Ki67+ proliferating cells or (F) γH2AX+ DNA-damaged cells in the CD44v6+ and CD44v6- fractions following radiation. g JAK2 expression was compared between the CD44v6+ and CD44v6- fractions in LoVo and patient-derived primary CRC cells. h and i To compare the stem cell frequencies between control cells and JAK2 knockdown cells, a limiting dilution assay was performed. j The primary tumors were resected from HCT116 xenograft mice as described in Fig. 2a. shCTRL- or shJAK2-transfected cancer cells were isolated from primary tumors and subjected to the LDA (12 wells/group). The bar graph represents the mean ± SD (n = 3). Statistical analyses were implemented using one-way ANOVA followed by Dunnett’s multiple comparison test against the control group or by Student’s t-test between two groups. The chi-squared test was used to compare the cell cycle distribution within different categories. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively