Fig. 1

a Validation of PTK7 expression levels in different cancer cell lines by Western blots. b Flow cytometry assay of the binding of SAC, CSAC, sgc8c control and sgc8c (250 nM, Cy5-labeled sequences). c Flow cytometry analysis of binding affinity of SAC to PTK7-positive cell lines, CEM and HCT116. d Confocal microscopy shows that SAC (250 nM, Cy5-labeled) could selectively internalize into PTK7- positive CEM and HCT116 cells after 2 h incubation at 37 °C. e Cell viability analysis by CCK8 assay (SAC, the red curves; artesunate, the blue curves). f The activation of SAC is related to Fe²⁺. The addition of exogenous FeSO₄ (10 μM) and iron chelator DFO (10 μM) were incubated with HCT116 cells for 2 h and then they were removed and SAC (10 μM) was added and incubated with HCT116 for 48 h. 10 μM DMSO, erastin and artesunate were added as control groups. ***P < 0.001, *P < 0.02; n = 4. g Measurement of the generation of reactive oxygen species (ROS) triggered by SAC (50, 100, 500 nM respectively): Cellular ROS levels were measured by dichlorofluorescein diacetate (DCFH-DA) (200 μM Fe²⁺, 200 μM DFO respectively, 5 μM DCFH-DA) after 24 h incubation with SAC. h The distribution of Cy5-labeled SAC (50 μM, 100 μL) and CSAC (50 μM, 100 μL) in tumor and major organs of HCT116 and K562 tumor-bearing nude mice after intravenous injection at 6 h visualized by Lumina XR in vivo imaging system