Fig. 4.

The phosphorylation of PTK6 promotes CRC cell stemness. A Western blot analyses were performed to verify the successful construction of FLAG-tagged empty vector, wild-type (WT), kinase-dead (KM) and constitutively active (YF) PTK6 recombinant mutants overexpression CRC cells. B In vitro limiting dilution assays show the effects of phosphorylated PTK6 on the formation of CSC spheres (mean ± SD, n = 12), likelihood ratio test. C Tumor sphere formation assays were performed to detect the sphere-forming ability of vector, PTK6^WT, PTK6^KM and PTK6^YF overexpression CRC cells (mean ± SD, n = 3). D Real-time q-PCR analysis shows the effects of phosphorylated PTK6 on the expression of stem cell markers  in CRC cells (mean ± SD, n = 3). E Western blot assays were performed to detect the effects of phosphorylated PTK6 on the expression of stem cell markers in CRC cells. F The number of CD133 + or SOX2 + cells was evaluated in the vector, PTK6^WT, PTK6^KM and PTK6^YF overexpression CRC cells by flow cytometry (mean ± SD, n = 3). G Co-immunofluorescent staining of PTK6 (red), CD133 (green) and EPACAM (green) in CRC cells. Scale bar represents 5 μm. H Co-immunofluorescent staining of PTK6 (red), CD133 (green) and EPACAM (green) in CRC tumor spheres. Scale bar = 5 μm. I In vivo limiting dilution analysis was performed to detect the tumorigenicity of vector, PTK6^WT, PTK6^KM and PTK6^YF overexpression CRC cells in nude mice (mean ± SD, n = 7). J IHC staining was used to detect the expression of PY342, SOX2 and Ki67 in indicated subcutaneous tumors of nude mice. Scale bar represents 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ^# indicates P > 0.05