Fig 2: Functional analysis of BaF3-ROR1, BaF3-ROR2, and BaF3-PTK7 clones. a Migration assay of BaF3 clones toward CXCL12 chemokine for 6 h. The graph shows the relative number of migrated cells normalized to BaF3 parental migrated cells. Data are represented as mean ± standard deviation (SD) and statistical comparison was performed with Student’s t test (n = 3). b Relative survival of BaF3 cells cultured in starvation media (no serum and no IL-3) in the presence or absence of Wnt5a (100 ng/ml) for 24 h. The graph shows mean ± SD (n = 3). In a and b, the statistical significance is indicated as *p < 0.05 and ***p < 0.001. c Immunoblotting analysis of the intracellular signaling in BaF3 parental cells and BaF3 clones expressing ROR1, ROR2, or PTK7. β-tubulin was used as a loading control. d Immunoblotting analysis of the cytoplasmic and nuclear lysates of BaF3-ROR1i clones that acquired IL-3 independence. Antibodies against the effectors of the pro-survival signaling were used to detect the protein levels and β-tubulin was used as a loading control for the cytoplasmic lysates, whereas PARP was used as a loading control for the nuclear lysates. e The relative proliferation of BaF3 parental and BaF3-ROR1i clone stimulated with 100 ng/ml Wnt5a or Wnt16b. Proliferation was measured at the indicated time points and the values were normalized to non-stimulated cells (set as value 1). The graph shows the mean ± SD (n = 3). Statistical comparison between the non-stimulated and Wnt-stimulated BaF3-ROR1i cells was done using Student’s t test as *p < 0.05

Fig 3: PTK7 associates with extracellular matrix organization and migration in breast cancer cells. (A) Gene Ontology (GO) was performed using DAVID: Functional Annotation Tools (https://david.ncifcrf.gov/tools.jsp) and Biological Process (BP) of PTK7 positively- and negatively-correlated genes were shown. (B) Pair-wise gene correlation of PTK7 with COL1A1, FN1, WNT5B, MMP11, MMP14 or SDC1 in breast invasive carcinoma were analyzed using GEPIA Correlation Analysis tools (http://gepia.cancer-pku.cn/detail.php?clicktag=correlation). (C) MDA-MB-231 and MDA-MB-468 cells were transduced with shNC, shPTK71 or shPTK72. Cells were stained for F-actin with TRITC-phalloidin. Pictures show the TRITC-tagged Phalloidin (red) and DAPI (purple). Presentative images are shown. Magnification, 400×; scale bars, 50 μm.

Fig 4: Simultaneous degradation of PTK7 and Met by the bi‐specific multitarget AptLYTACs. A) Western blot result of dual‐targeted degradation by bi‐specific AptLYTACs (Sgc8:SL1 = 2:1, SA:aptamer = 1:3) after treatment without AptLYTACs (NC) or with different concentrations of bpM6P‐Sgc8‐SL1‐SA for 4 h in SUM159 cells. B) PTK7 and Met expression in SUM159 cells after treatment with bpM6P‐Sgc8‐SL1‐SA (Sgc8:SL1 = 2:1) for different periods of time as indicated. C) Western blot result of SUM159 cells after being treated without (NC) or with bi‐specific AptLYTACs with different aptamer loading ratios (Sgc8:SL1:SA = 1:2:1, 2:1:1, and 4:2:1) for 4 h. D) Quantitative result of grayscale analysis of (C).

Fig 5: PTK7‐targeted protein degradation by monovalent and multivalent AptLYTACs. A) Western blot result of PTK7 levels in Jurkat cells treated with 500 nm of AptLYTACs with different ratios of SA:Sgc8 (1:1, 1:3, 1:5, and 1:7) for 4 h. B,C) Western blot analysis of PTK7 after treatment with bpM6P‐Sgc8‐SA (SA:Sgc8 = 1:1) or bpM6P‐Sgc8‐SA (SA:Sgc8 = 1:3) in various concentrations for 2 h in Jurkat cells. D) Binding affinities of FAM‐labeled Sgc8 and FAM‐AptLYTACs (SA:Sgc8 = 1:1, 1:3, 1:5, or 1:7) toward Jurkat cells, as measured by flow cytometry. Data were shown as mean ± SD obtained from three independent experiments. E) Fluorescence intensities of Jurkat cells treated with 200 nm FAM‐Sgc8 or different FAM‐labeled AptLYTACs (SA:Sgc8 = 1:1, 1:3, 1:5, and 1:7) for 0.5 h at room temperature, as measured by flow cytometry. F) Native PAGE analysis of the SA‐Sgc8 (1:3, 1:7) assembled with an equal amount of bpM6P. G) Fluorescence intensities of Ramos cells receiving the same treatments as described in (E). H,I) Western blot analysis of the degradation kinetics of PTK7 after treatment with 500 nm bpM6P‐Sgc8‐SA (SA:Sgc8 = 1:3) for different time periods (1–12 h) in Jurkat cells and SUM159 cells. J) Western blot analysis of PTK7 in Jurkat cells after treatment with bpM6P‐Sgc8‐SA (SA:Sgc8 = 1:3) at various aptamer concentrations (10–1000 nm) for 4 h.