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. 2022 Oct 4;15(1):13–37. doi: 10.1016/j.jcmgh.2022.09.015

Figure 6.

Figure 6

The enrichment of PTK7 in HCC is responsive to the activation of TGF-β signaling and potentially mediated by stimulated SOX9 activity. (A) Patients in the TCGA-LIHC cohort were ranked according to the transcriptomic level of PTK7 in their tumors. Publicly available RNA sequencing data of the 25% highest-ranking patients were subjected to GSEA to identify molecular signatures and pathways deregulated under enriched PTK7 conditions compared with the 25% lowest-ranking patients. TGF-β signaling, which is highly relevant to metastasis, was identified as one of the top pathways significantly enriched under high-PTK7 conditions. The top 10 deregulated pathways under the high-PTK7 condition are arranged in a bubble diagram for reference. (B) Heatmap showing the correlation of the tumor PTK7 level with 18 TGF-β signaling signature genes in all of the paired tumor/nontumor (T/NT) cases of the TCGA-LIHC cohort. The T/NT fold change of all signature genes was calculated, and their activation status for each HCC case was based on a numeric scoring system, where a score of 2 means the most activated and a score of -2 is the most inactivated. A TGF-β score was assigned to each patient by integrating the scores of all 18 signature genes. An overall zero or negative TGF-β score was representative of a TGF-β–suppressive environment, and a positive overall score was representative of a TGF-β–activated environment. PTK7 expression was compared between HCC samples with a positive or negative TGF-β score using the Student t test. (C) Western blot validation of dose-dependent up-regulation of PTK7 and SOX9 expression in PLC/PRF/5 cells after a single treatment of recombinant TGF-β1 for 24 hours at various doses. Quantification of PTK7 and SOX9 expression was performed in ImageJ (National Institutes of Health, Bethesda, MD). (D) Western blot validation of PTK7 and SOX9 expression in PLC/PRF/5 cells after TGF-β1 single treatment or in combination with a TGF-βR1–specific inhibitor, LY-364947, for 24 hours. (E) Representative images of immunocytochemistry targeting SOX9 in PLC/PRF/5 cells after TGF-β1 single treatment or in combination with LY-364947 (Combo) for 24 hours. 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to locate the nuclei of stained cells. The average nuclear localization of SOX9 was quantified in ImageJ and compared between treatment groups using the Student t test. (F) Western blot validation of PTK7 levels in SOX9-knockdown PLC/PRF/5 cells after TGF-β1 single treatment. DMSO, dimethyl sulfoxide; DN, down-regulated; FDR, false discovery rate; IF, immunofluorescence; mRNA, messenger RNA; NTC, nontarget control. P values (∗P < .05, ∗∗P < .01, ∗∗∗P < .001).