Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: PTK7 was the most significantly deregulated kinase in metastatic HCC and was enriched in advanced HCC tumors. ( A ) A total of 509 commonly deregulated kinases in cancer were extracted from the nCounter Human Kinase Panel Gene List, and their expression pattern was examined in the TCGA-LIHC patient cohort. Thirty-five candidate kinases were overexpressed in tumor and advanced HCC, and ranked (from top to bottom) according to their extent of up-regulation and evaluated against the GSE45114 data set, which described transcriptomic profiles of nontumor and tumor tissues harvested from metastatic HCC samples and their nonmetastatic counterparts. The heatmap was constructed to represent the fold-change in the expression of each candidate kinase in HCC tumors compared with nontumor samples under a metastatic or nonmetastatic background. Only 3 kinases (TTK, PTK7, and CDC7, marked with an asterisk ) were significantly overexpressed in metastatic HCC, and PTK7 was the most significantly deregulated. ( B ) Statistical evaluation of the fold change in PTK7 expression (tumor vs nontumor) for 228 pairs of HCC samples evenly grouped by their predicted risk of metastasis as described in the GSE14520 cohort by paired t test between each tumor/nontumor comparison. The predicted risk of metastasis was defined by the integral signature score of 161 metastatic genes that robustly correlated with recurrence and adverse clinical outcome. ( C ) The distribution of early and advanced HCC cases in the high-PTK7 and low-PTK7 samples of the TCGA-LIHC data set compared using the chi-squared test. ( D ) The overall survival distribution of patients with high or low PTK7 expression in the TCGA-LIHC data set compared using the Gehan–Breslow–Wilcoxon test. ( E ) Representative images of high-PTK7 staining from IHC on TMA containing tumor and nontumor samples of HCC patients. Scale bar : 100 μm. ( F ) The distribution of high- and low-PTK7 cases in tumor and nontumor samples of TMA compared using the chi-squared test. ( G ) The overall survival distribution of TMA patients with high or low PTK7 expression compared using the log-rank test. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Expressing, Construct, Comparison, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: Up-regulation of PTK7 in metastatic HCC. ( A ) The evaluation of PTK7 expression in metastatic vs nonmetastatic HCC samples in GSE45114, by Student t test. ( B ) The expression of PTK7 in primary tumor and portal vein tumor thrombus (PVTT) tissue of HCC patients was evaluated against that of paired normal tissue using the GSE77509 data set, by paired t test. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: The expression level and transcriptomic activity of SOX9 are clinically and functionally correlated with PTK7. ( A ) A total of 133 TFs potentially binding to the promoter region of PTK7 , proximal to the transcriptional start site (between -1000 and +1), were compiled from the online Gene Transcription Regulation Database. Fourteen candidate TFs that highly correlated with PTK7 ( r > 0.3, determined by Pearson correlation) and were overexpressed in tumor HCC at the messenger RNA (mRNA) level (analyzed using TCGA-LIHC data as a normalized count) were examined further for their specific enrichment in metastatic HCC using GSE45114 data. Only SOX9, nuclear respiratory factor 1 (NRF1), and TEA domain transcription factor (TEAD4) satisfied all of the tested criteria. The correlation of SOX9 and PTK7 expression at the mRNA level is shown for reference. ( B ) IHC was performed on TMA to investigate the extent of SOX9 nuclear localization in tumor HCC samples in which cases were designated as SOX9 high (>75% nuclei), medium (25%–75% nuclei), or low (<25% nuclei), depending on the amount of tumor cells showing apparent SOX9 nuclear staining. Graded expression of SOX9 was compared with PTK7 in the same TMA. Representative images of IHC are shown. Scale bar : 100 μm. ( C ) The distribution of cases with various nuclear SOX9 expression in the high-PTK7 and low-PTK7 samples of TMA was compared using the chi-squared test. ( D ) Two truncated versions of the PTK7 promoter region, one containing the predicted SOX9 binding site (T1) and one without (T2), were expressed in HCC cell lines via transient transfection. The ability of the promoter elements to drive protein expression was quantified using the Dual-Glo luciferase reporter assay and compared using the Student t test. ( E ) A ChIP assay was performed in HCC cell lines to examine the physical binding of SOX9 to the PTK7 promoter region. Targeted enrichment of the predicted binding sequence was quantified using reverse-transcription quantitative PCR and compared with mouse IgG immunoprecipitation control (IgG). ( F ) The messenger RNA (mRNA) levels of PTK7 and SOX9 were quantified and validated in HCC cells after successful knockdown of SOX9 by short hairpin RNA (shRNA). ( G ) Western blot validation of PTK7 suppression after SOX9 depletion by shRNA knockdown. ( H ) Two truncated PTK7 promoter elements, one harboring the predicted SOX9-binding motif (T1) and the other lacking the same motif (T2), were transfected into SOX9-knockdown (KD) cells and nontarget control (NTC) cells. The resultant luciferase signal induced by the 2 elements in the different cell lines was compared using the Student t test. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Expressing, Activity Assay, Binding Assay, Staining, Transfection, Luciferase, Reporter Assay, Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunoprecipitation, Control, Knockdown, shRNA, Western Blot, Biomarker Discovery

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: SOX9 as an important upstream regulator of PTK7 expression. ( A ) The correlation of the fold change (FC) in PTK7 and SOX9 expression of HCC tumor compared with nontumor tissue, analyzed using data from GSE45114. ( B ) Same statistical evaluation of GSE14520 data set as described in Figure 1 B for the fold change in SOX9 expression of paired tumor/nontumor HCC samples with different predicted risk of metastasis, by paired t test. ( C ) The distribution of high-, medium-, and low-SOX9 cases in tumor or nontumor samples of TMA, compared by chi-squared test. ( D ) The overall survival distribution of TMA patients grouped according to their tumor PTK7 and SOX9 level, compared by log-rank test. ( E ) The frequency matrix of the DNA-binding motif of SOX9 as reported in JASPAR database. A single SOX9 binding site was predicted on the PTK7 promoter between the -450 and -460 region upstream of transcription start site. ( F ) Two truncations of the proximal PTK7 promoter region were synthesized and integrated into the pGL3-basic vector backbone for luciferase reporter assay. T1 truncation contained the predicted SOX9 binding site while T2 truncation did not. ( G ) The ability in driving protein expression of the 2 truncated promoter elements of PTK7 was quantified by the Dual-Glo luciferase reporter assay after transient transfection and compared by Student t test. ( H ) The physical binding of SOX9 to the PTK7 promoter region was examined by ChIP assay. Targeted enrichment of the predicted binding sequence was quantified by quantitative PCR and compared with mouse IgG immunoprecipitation control (IgG). ( I ) The messenger RNA (mRNA) level of PTK7 and SOX9 was quantified and validated in Hep3B cells after successful knockdown of SOX9 by short hairpin RNA (shRNA). ( J ) Western blot validation of the suppression of PTK7 level after SOX9 depletion by shRNA knockdown in Hep3B. H, high; L, low; M, medium; NTC, nontarget control. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Expressing, Binding Assay, Synthesized, Plasmid Preparation, Luciferase, Reporter Assay, Transfection, Sequencing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Control, Knockdown, shRNA, Western Blot, Biomarker Discovery

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: Expression of PTK7 and SOX9 in HCC cell lines. ( A ) A comparison of the expression of endogenous PTK7, SOX9, and secretory PTK7 in a panel of HCC cell lines. Coomassie Blue staining was used as a loading control for total secretory protein. ( B ) Quantitative PCR analysis on PTK7 messenger RNA (mRNA) level relative to that of the housekeeping gene β-actin across the HCC cell line panel. ( C ) Flow cytometry performed to evaluate the membrane expression of PTK7 across the HCC cell line panel. A mouse APC-conjugated IgG2a isotype control was used as the negative control. ( D ) A heterogenous population of PTK7-expressing and nonexpressing cells in PLC/PRF/5 as determined by flow cytometry. The 20% subsets with the highest- or lowest-membrane PTK7 expression were sorted out for functional and molecular assays. FSC, forward scatter.

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Expressing, Comparison, Staining, Control, Real-time Polymerase Chain Reaction, Flow Cytometry, Membrane, Negative Control, Functional Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: 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).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Activation Assay, Activity Assay, RNA Sequencing, Expressing, Western Blot, Biomarker Discovery, Recombinant, Immunocytochemistry, Staining, Knockdown, Immunofluorescence, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: PTK7 enrichment in HCC tumor is not driven by and does not result in the activation of Wnt signaling. ( A ) A bioinformatic analysis was performed on TCGA-LIHC cohort to compare the transcriptomic level of PTK7 under HCC driven by different CTNNB1 status. Groups were compared by Student t test. ( B ) IHC was performed on formalin-fixed, paraffin-embedded tissue of terminal livers dissected from C57BL/6 mice transformed with a ΔN90-CTNNB1/c-MYC driver plasmid by hydrodynamic tail-vein injection. Representative IHC images showing PTK7 and β-catenin expression pattern at tumor boundaries. ( C ) The subcellular localization of β-catenin was investigated in MHCC97L cells overexpressing PTK7 by subcellular fractionation and Western blot. ( D ) The transcriptional activity of β-catenin in Wnt3a-stimulated MHCC97L cells overexpressing PTK7 was examined by TOP/FOP assay and compared with treated EV control by Student t test. NOT, nontumor; OE, overexpression; T, tumor. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Activation Assay, Formalin-fixed Paraffin-Embedded, Transformation Assay, Plasmid Preparation, Injection, Expressing, Fractionation, Western Blot, Activity Assay, Control, Over Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: In vitro analyses of TGF-β1–treated HCC cells. ( A ) PTK7 correlation with 6 exhausted T-cell signature markers (HAVCR2, TIGIT, LAG3, PDCD1, CXCL13, and LAYN) was examined by Pearson correlation using TCGA-LIHC data set and aided by the publicly available GEPIA2 analysis platform with all expression in terms of transcript per million. ( B ) Statistical comparison of the expression of TGFB1 / 2 / 3 in metastatic and nonmetastatic HCC by GSE45114 and the Student t test. ( C ) Same statistical evaluation of the GSE14520 data set as described in Figure 1 B for the fold change in TGFB1 expression of paired tumor/nontumor HCC samples with different predicted risk of metastasis, by paired t test. ( D ) Representative images and the quantification of migrating cells by Transwell assay after TGF-β1 single treatment of 24 hours’ duration. Statistical comparisons by the Student t test. ( E ) Western blot validation of dosage-dependent up-regulation of PTK7 and SOX9 expression in Hep3B cells after a single treatment of recombinant TGF-β1 for a duration of 24 hours at various dosages. Quantification of PTK7 and SOX9 expression was performed by ImageJ. ( F ) Western blot validation of PTK7 and SOX9 expression in Hep3B cells after TGF-β1 single treatment or in combination with LY-364947, a TGF-βR1–specific inhibitor, for a duration of 24 hours. ( G ) Representative images of immunocytochemistry targeting SOX9 in Hep3B cells after TGF-β1 single treatment or in combination with LY-364947 (Combo) for a duration of 24 hours. 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to locate the nuclei of stained cells. The average nuclear localization of the SOX9 was quantified by ImageJ and compared between treatment groups by the Student t test. DMSO, dimethyl sulfoxide. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: In Vitro, Expressing, Comparison, Transwell Assay, Western Blot, Biomarker Discovery, Recombinant, Immunocytochemistry, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: PTK7 enrichment and overexpression lead to more aggressive metastasis of HCC cells, and PTK7 knockout reduced the metastatic capacity of cells, and the prometastatic properties of PTK7 potentially are mediated by its secretory domain. ( A ) Low- and high-PTK7–expressing cells were isolated from the HCC cell line PLC/PRF/5 using FACS, and the separation was validated by Western blot. Sorted PLC/PRF/5 cells were subjected to Transwell migration and invasion assays. The number of migrating or invading cells was compared using the Student t test. ( B ) CRISPR-Cas9–mediated knockout of PTK7 was performed in Hep3B cells, followed by Western blot validation and Transwell migration and invasion assays. The number of migrating or invading cells was compared with the transfection control (Ctrl) using 1-way analysis of variance. ( C ) PTK7 was overexpressed in MHCC97L cells and validated using Western blot. The number of overexpressing cells migrating or invading in Transwell assays was compared with EV control using the Student t test. ( D ) CRISPR-Cas9–mediated knockout of Ptk7 was performed on murine RIL175 cells, followed by Western blot validation and Transwell migration and invasion assays. The number of migrating or invading cells was compared with the transfection control (Ctrl) using 1-way analysis of variance. All representative images of Transwell assays were taken under a light microscope at 40× magnification with at least 4 microscopic fields captured for statistical analyses. Scale bars : 100 μm. KO, knockout; OE, overexpression. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Over Expression, Knock-Out, Expressing, Isolation, Western Blot, Migration, CRISPR, Biomarker Discovery, Transfection, Control, Light Microscopy

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: PTK7 in vitro functional assays performed on various HCC cell lines. ( A ) PTK7 was overexpressed in MHCC97L cells and subsequently suppressed by lentiviral-based knockdown using 2 independent short hairpin RNA (shRNA) structures (sh845 and sh923). The number of cells migrating or invading in Transwell assays were compared with EV + nontarget control (NTC) by 1-way analysis of variance with multiple t tests. The successful knockdown of PTK7 was validated by Western blot. ( B ) Representative images and the comparison of the number of foci formed by sorted PLC cells and MHCC97L cells overexpressing PTK7. Statistical comparisons were performed by the Student t test. ( C ) The predicted tumor-initiating cell frequency of sorted PLC cells and MHCC97L overexpression cells was determined by extreme limiting dilution assay and analyzed with a publicly available algorithm. The frequency was represented by the estimated population of cells required for the detection of 1 tumor-initiating cell. ( D ) Representative H&E staining of a lung tissue section harvested from mice injected with RIL175 through the tail vein. Metastases are indicated with black arrows . Ctrl, control; OE, overexpression. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: In Vitro, Functional Assay, Knockdown, shRNA, Control, Western Blot, Comparison, Over Expression, Limiting Dilution Assay, Staining, Injection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: PTK7 overexpression promotes extrahepatic metastasis to the lung of liver-engrafted HCC cells, and PTK7 knockout suppresses the ability of cells to extravasate into the lung. ( A ) In vivo detection of luciferase-induced luminescence of tumor nodules and images of livers dissected from nude mice 8 weeks after orthotopic liver injection of MHCC97L overexpressing cells. ( B ) External morphology of HCC nodules formed in the livers of nude mice 8 weeks after orthotopic liver injection of MHCC97L-overexpressing cells. ( C ) H&E-stained tissue sections of lungs dissected from the orthotopic injection model showing the formation of lung metastases (indicated by white arrows). The number of discernible metastases formed in the lungs of mice injected with MHCC97L-overexpressing cells was counted and compared using the Student t test. ( D ) H&E-stained tissue sections of livers dissected from the orthotopic injection model showing different features of the tumor boundary. The presence of microsatellites is indicated by black arrows . ( E ) A summary table of the parameters related to metastatic aggressiveness of the tumors in the orthotopic injection model. ( F ) Murine RIL175 cells with Ptk7 depleted by CRISPR-Cas9–mediated knockout were injected into nude mice through the tail vein to observe lung metastasis formation. Luciferase-induced luminescence of tumor nodules developed from RIL175 cells was detected using in vivo imaging 7 days after tail vein injection. The total detectable radiance was compared between the control and Ptk7-knockout groups using 1-way analysis of variance. Ctrl, control; OE, overexpression. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Over Expression, Knock-Out, In Vivo, Luciferase, Injection, Staining, CRISPR, In Vivo Imaging, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: The up-regulation of PTK7 resulted in enrichment of the EMT components SLUG and ZEB1, which may confer the prometastatic properties of PTK7. ( A ) RNA sequencing was performed on HCC cells with differential PTK7 expression, and Hep3B control was compared against one of the PTK7-knockout clones. FACS-sorted PLC/PRF/5 cells were compared against each other. Commonly deregulated genes (total, 1936 genes) were identified in the 2 comparison groups with a threshold of transcript per million greater than 1 and relative fold-change difference of 1.2 or greater, and then subjected to GSEA. EMT was one of the top enriched pathways under PTK7 enrichment. ( B ) The proteomic levels of SLUG and ZEB1 in PTK7-overexpressing MHCC97L cells, sorted PLC/PRF/5 cells, and PTK7-knockout Hep3B cells were evaluated using Western blot. ( C ) Representative images of immunocytochemistry (ICC) targeting SLUG and ZEB1 in PTK7-overexpressing MHCC97L cells. 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to locate the nuclei of stained cells. The average nuclear localization of SLUG and ZEB1 was quantified in ImageJ and compared between EV and overexpressing cells using the Student t test. ( D ) IHC was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections derived from the livers of 14 HCC patients to detect SLUG and ZEB1 expression. Representative images of tumors with high or low PTK7 expression are shown. The proportion of HCC nuclei observed with SLUG expression was evaluated further using light microscopy and compared between PTK7-low and PTK7-high patient groups with at least 4 microscopic fields captured for statistical analyses. High-SLUG nuclear expression was defined by >50% nuclei observed with intense DAB staining, and vice versa. Group comparisons were performed using the Fisher exact test. ( E ) IHC was performed on FFPE tissue sections of liver tumors developed after orthotopic injection of MHCC97L cells to compare E-cadherin, SLUG, and ZEB1 expression patterns between EV control and PTK7-overexpressing xenografts. Scale bars : 100 μm. Ctrl, control; IF, immunofluorescence. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: RNA Sequencing, Expressing, Control, Knock-Out, Clone Assay, Comparison, Western Blot, Immunocytochemistry, Staining, Formalin-fixed Paraffin-Embedded, Derivative Assay, Light Microscopy, Injection, Immunofluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: RNA sequencing and representative images of IHC conducted on lung sections harvested from the orthotopic injection mice model. ( A ) A bubble plot showing the top 10 enriched Hallmark pathways under PTK7-enriched conditions as determined by GSEA analysis. ( B ) Representative images of immunocytochemistry (ICC) targeting SLUG and ZEB1 in PLC/PRF/5 cells sorted by their PTK7 expression. 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to locate the nucleus of stained cells. The average nuclear localization of the SLUG and ZEB1 was quantified by ImageJ and compared between PTK7 -ve and PTK7 +ve cells by the Student t test. ( C ) Representative images of ICC targeting SLUG in MHCC97L, of which PTK7 was overexpressed and subsequently suppressed by lentiviral-based knockdown using 2 independent short hairpin RNA structures (sh845 and sh923). DAPI staining was used to locate the nucleus of stained cells. The average nuclear localization of SLUG was quantified by ImageJ and compared between the 4 transfected lines by 1-way analysis of variance with multiple t tests. ( D ) Images showing IHC staining of PTK7 and SLUG performed on lung metastases resulted from orthotopic liver injection of MHCC97L cells with or without PTK7 overexpression. DN, down-regulated; E2F, E2F transcription factor; FDR, false discovery rate; G2M, G2 to M phase cell cycle transition; IF, immunofluorecence; IL, interleukin; NTC, nontarget control; OE, overexpression; STAT5, signal transducer and activator of transcription 5. P values (∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001).

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: RNA Sequencing, Injection, Immunocytochemistry, Expressing, Staining, Knockdown, shRNA, Transfection, Immunohistochemistry, Over Expression, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Protein Tyrosine Kinase 7 (PTK7) Promotes Metastasis in Hepatocellular Carcinoma via SOX9 Regulation and TGF-β Signaling

doi: 10.1016/j.jcmgh.2022.09.015

Figure Lengend Snippet: Primers Used in This Study

Article Snippet: Marking of surface PTK7 was performed using an APC-conjugated anti-human PTK7 mouse antibody (130-099-660; Miltenyi Biotec) with an allophycocyanin (APC)-conjugated mouse IgG2a (130-113-831; Miltenyi Biotec) stained as negative control.

Techniques: Sequencing

Journal: Signal Transduction and Targeted Therapy

Article Title: An aptamer-drug conjugate for promising cancer therapy with comprehensive evaluation from rodents to non-human primates

doi: 10.1038/s41392-025-02399-1

Figure Lengend Snippet: Characterization of Sgc8c-M in synthesis, targeting, internalization, and cytotoxicity in vitro. a Chemical structure, HPLC, and mass spectrum of Sgc8c-M. b Surface plasmon resonance (SPR) analysis of Sgc8c-M for binding to recombinant PTK7 proteins from humans, mice, rats, and cynomolgus monkeys at 400 nM. c Flow cytometry analysis of Cy5-labeled Ctrl, Sgc8c, Ctrl-M, and Sgc8c-M (250 nM) for binding to PTK7-positive SUM159 and MDA-MB-468 cells, as well as PTK7-negative Ramos cells. d Flow cytometry analysis of the internalization and endocytic pathway of Cy5-Sgc8c-M in SUM159 cells. The endocytic pathway was determined by measuring the uptake upon the application of various inhibitors. M-β-CD (0.2 mM), CPZ (25 μM), and AMI (100 μM) were preincubated with cells for 30 min at 37 °C; then 400 nM Cy5-Sgc8c-M were added and incubation continued for another 2 h ( n = 3). Unpaired t test was used in comparison to control, ** P < 0.01, *** P < 0.001, ns, not significant. e Confocal microscopy analysis of Cy5-Sgc8c-M (400 nM) for internalization in SUM159 cells at 0.5 h and 2 h. Scale bars, 10 μm. f Cytotoxicity of VcMMAE and Sgc8c-M in SUM159 and MDA-MB-468 cells after a 72-h incubation ( n = 3). Data are presented as mean ± SEM

Article Snippet: The primary antibody, anti-PTK7 rabbit polyclonal antibody (#A9839, abclonal), was diluted to a concentration of 1:250 and applied to the sections for overnight incubation at 4 °C.

Techniques: In Vitro, SPR Assay, Binding Assay, Recombinant, Flow Cytometry, Labeling, Incubation, Comparison, Control, Confocal Microscopy

Journal: Signal Transduction and Targeted Therapy

Article Title: An aptamer-drug conjugate for promising cancer therapy with comprehensive evaluation from rodents to non-human primates

doi: 10.1038/s41392-025-02399-1

Figure Lengend Snippet: In vivo antitumor effect of Sgc8c-M in TNBC. a IHC characterization of PTK7 expression on tumor tissues of SUM159, MDA-MB-468, and TNBC PDX models. Scale bars, 20 μm. b Tumor volume and body weight of SUM159 tumor-bearing mice from a dose screening trial of Sgc8c-M ( n = 4, one-way ANOVA). c , d , f Tumor growth curves for SUM159 ( n = 4), MDA-MB-468 ( n = 4, unpaired t test), and TNBC PDX ( n = 5, unpaired t test) tumor-bearing mice after multiple dosing of 7 mg/kg Sgc8c-M (intravenous injection, i.v.), 10 mg/kg paclitaxel (intraperitoneal injection, i.p.), or 0.359 mg/kg MMAE (i.v.). e , g Tumor-killing effect study of Sgc8c-M in MDA-MB-468 ( n = 3, unpaired t test) and TNBC PDX ( n = 3, unpaired t test). Mice were given 7 mg/kg Sgc8c-M intravenously from day 0, and immunohistochemical characterization of CK19, Ki67, and pHH3 was performed at 48–96 h (48 h for MDA-MB-468, 96 h for TNBC PDX) at the end of administration. Scale bars, 20 μm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: The primary antibody, anti-PTK7 rabbit polyclonal antibody (#A9839, abclonal), was diluted to a concentration of 1:250 and applied to the sections for overnight incubation at 4 °C.

Techniques: In Vivo, Expressing, Injection, Immunohistochemical staining

Journal: Signal Transduction and Targeted Therapy

Article Title: An aptamer-drug conjugate for promising cancer therapy with comprehensive evaluation from rodents to non-human primates

doi: 10.1038/s41392-025-02399-1

Figure Lengend Snippet: Antitumor effect of Sgc8c-M in pancreatic cancer. a Cytotoxicity of VcMMAE, Sgc8c-M, and MMAE in MIA PaCa-2 cells after a 72-h incubation ( n = 3). b , e , f Antitumor effects of Sgc8c-M for MIA PaCa-2, PDX F4, and PDX F5 in vivo ( n = 5, unpaired t test). Tumor-bearing mice were dosed by i.v. every four days for 5–6 cycles with 7 mg/kg Sgc8c-M or 0.359 mg/kg MMAE. PTK7 IHC characterization and hematoxylin-eosin (HE) staining on these tumors showed the differences in PTK7 expression and tissue structure. Scale bars, 20 μm. c MMAE concentration in MIA PaCa-2 tumors at 2 h after a single dose of 7 mg/kg Sgc8c-M or 0.359 mg/kg MMAE ( n = 3, unpaired t test). d Tumor-killing effect study of Sgc8c-M and MMAE in MIA PaCa-2 xenografts. Mice received 7 mg/kg Sgc8c-M or 0.359 mg/kg MMAE every four days for three cycles (q4d × 3) with Ki67 and pHH3 analyzed 24 h after the final dose ( n = 3, one-way ANOVA). Scale bars, 20 μm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: The primary antibody, anti-PTK7 rabbit polyclonal antibody (#A9839, abclonal), was diluted to a concentration of 1:250 and applied to the sections for overnight incubation at 4 °C.

Techniques: Incubation, In Vivo, Staining, Expressing, Concentration Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: An aptamer-drug conjugate for promising cancer therapy with comprehensive evaluation from rodents to non-human primates

doi: 10.1038/s41392-025-02399-1

Figure Lengend Snippet: Antitumor activity of Sgc8c-M in HT-29, NCI-H1975, and OVCAR3 xenograft models. In vivo therapeutic efficacy of Sgc8c-M in HT-29 ( n = 4, one-way ANOVA) ( a ), NCI-H1975 ( n = 3) ( b ), and OVCAR3 ( n = 3, one-way ANOVA) ( c ) xenografts. Tumor-bearing mice were dosed with 7 mg/kg Sgc8c-M (i.v.) or 10 mg/kg paclitaxel (i.p.) when the tumor size reached an average of 100–200 mm 3 . Histology and PTK7 expression in these models were shown as HE staining and IHC staining on tumor tissues of untreated mice, respectively. d The antitumor effects of Sgc8c-M (3.5 mg/kg) and PTK7-targeted ADC h6M24-VcMMAE (DAR4, 5 mg/kg) in the OVCAR3 model ( n = 5, unpaired t test). Scale bars, 20 μm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01

Article Snippet: The primary antibody, anti-PTK7 rabbit polyclonal antibody (#A9839, abclonal), was diluted to a concentration of 1:250 and applied to the sections for overnight incubation at 4 °C.

Techniques: Activity Assay, In Vivo, Drug discovery, Expressing, Staining, Immunohistochemistry

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide

doi: 10.1016/j.mcpro.2021.100144

Figure Lengend Snippet: Potential CADA-affected SILAC targets selected for validation

Article Snippet: The endoplasmic reticulum lectin 1 (ERLEC1) expression vector (pGEM-T backbone) was purchased from Sinobiological, the PTK7 expression vector (pDONR223 backbone) from Addgene, and the DNAJ homolog subfamily C member 3 (DNAJC3) expression vector (pDONR223 backbone) from DNASU plasmid repository.

Techniques: Multiplex sample analysis, Immunopeptidomics, Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide

doi: 10.1016/j.mcpro.2021.100144

Figure Lengend Snippet: CADA downmodulates cell-surface huCD4 and PTK7 in lymphoid cells and the transient expression of huCD4, SORT, PTK7, ERLEC1, and DNAJC3 in transfected cells. A , concentration–response curves of CADA for huCD4 and PTK7 in SUP-T1 cells. Cells were incubated with CADA for 24 h. Tested CADA concentrations were 50, 10, 2, 0.4, 0.08, and 0.016 μM. Expression of the surface receptors was measured with flow cytometry and normalized to the DMSO control. A four parameter concentration response curve was fitted to data from at least three replicate experiments. Values are mean ± SD; n ≥ 3. B , Western blot images of cell lysates from nontransfected (NT) and huCD4-V5, SORT-V5, PTK7-V5, ERLEC1-V5, or DNAJC3-V5 transfected HEK293T cells treated for 24 h with different CADA concentrations. Protein bands were visualized using an antibody against the V5 tag, whereas for the cell loading control, an anticlathrin or anti–β-actin antibody was used. One representative experiment out of 3 to 5 is shown. C , concentration–response curves of CADA for huCD4, SORT, PTK7, ERLEC1, and DNAJC3 in transfected HEK293T cells. Samples from ( B ) were quantified and normalized to the clathrin (huCD4, ERLEC1, and DNAJC3) or β-actin (SORT and PTK7) internal control. A four parameter concentration response curve was fitted to the data from at least three replicate experiments. Values are mean ± SD; n ≥ 3. CADA, cyclotriazadisulfonamide; DMSO, dimethyl sulfoxide; DNAJC3, DnaJ homolog subfamily C member 3; ERLEC1, endoplasmic reticulum lectin 1; HEK293T, human embryonic kidney 293T cells; huCD4, human CD4; PTK7, inactive tyrosine-protein kinase 7; SORT, sortilin.

Article Snippet: The endoplasmic reticulum lectin 1 (ERLEC1) expression vector (pGEM-T backbone) was purchased from Sinobiological, the PTK7 expression vector (pDONR223 backbone) from Addgene, and the DNAJ homolog subfamily C member 3 (DNAJC3) expression vector (pDONR223 backbone) from DNASU plasmid repository.

Techniques: Expressing, Transfection, Concentration Assay, Incubation, Flow Cytometry, Control, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide

doi: 10.1016/j.mcpro.2021.100144

Figure Lengend Snippet: SILAC hits differentially respond to CADA in transfected cells. A , schematic representation of the expected mRNA and protein products of the tGFP-P2A-BFP construct. The constructs express huCD4 that is anchored in the plasma membrane via its transmembrane domain and with tGFP at the cytosolic tail. As the SP is cleaved by the ER lumenal signal peptidase during protein biogenesis, the mature huCD4 variants differ only in their N terminus (62 residues). B , four parameter concentration–response curves for CADA of huCD4, SORT, PTK7, ERLEC1, and DNAJC3 cloned in the same tGFP-P2A-BFP plasmid backbone as shown in ( A ). HEK293T cells were transiently transfected with the tGFP-P2A-BFP constructs and incubated with different CADA concentrations for 24 h. Protein levels in CADA-treated samples are normalized to the DMSO control (set at 1.0). Curves are fitted to data from 3 to 4 replicate experiments. Values are mean ± SD; n ≥ 3. BFP, blue fluorescent protein; CADA, cyclotriazadisulfonamide; DMSO, dimethyl sulfoxide; DNAJC3, DnaJ homolog subfamily C member 3; ER, endoplasmic reticulum; ERLEC1, endoplasmic reticulum lectin 1; HEK293T, human embryonic kidney 293T cells; huCD4, human CD4; PTK7, inactive tyrosine-protein kinase 7; SILAC, stable isotope labeling by amino acids in cell culture; SORT, sortilin; SP, signal peptide; tGFP, turbo GFP.

Article Snippet: The endoplasmic reticulum lectin 1 (ERLEC1) expression vector (pGEM-T backbone) was purchased from Sinobiological, the PTK7 expression vector (pDONR223 backbone) from Addgene, and the DNAJ homolog subfamily C member 3 (DNAJC3) expression vector (pDONR223 backbone) from DNASU plasmid repository.

Techniques: Multiplex sample analysis, Transfection, Construct, Clinical Proteomics, Membrane, Concentration Assay, Clone Assay, Plasmid Preparation, Incubation, Control, Quantitative Proteomics, Cell Culture

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide

doi: 10.1016/j.mcpro.2021.100144

Figure Lengend Snippet: CADA diverts the nonglycosylated preprotein for proteasomal degradation. A , autoradiogram of cell lysates from nontransfected (NT) and huCD4-V5 transfected CHO-K1 cells pulsed for 30 min with [ 35 S]methionine/cysteine. Pulsed-labeled cells were then chased for 45 min in the presence or the absence of CADA (10 μM) and/or MG132 (200 nM). Open arrowhead represents the translocated (and glycosylated) huCD4 mature protein fraction, and solid arrowhead represents the nonglycosylated huCD4 preprotein. Graph on the right shows the quantification of the presented radioblot. For each sample, the fraction of preprotein and mature protein is calculated and given as percentage of total amount of protein. B , Western blot images of cell lysates from nontransfected (NT) and huCD4-V5, SORT-V5, and PTK7-V5 transfected HEK293T cells treated for 24 h with different CADA concentrations and 200 nM MG132. Protein bands were visualized using an antibody against the V5 tag, whereas anticlathrin (huCD4) or anti-β-actin (SORT and PTK7) antibody was used for the cell loading controls. Do note that the combination of MG132 with 10 or 2 μM CADA strongly reduces the level of mature protein, presumably by blocking the residual translocation of substrate that is normally seen with CADA. C , same as in ( B ) but for 10 μM CADA and/or 200 nM MG132. Lysates were treated with endo H prior to SDS-PAGE and Western blotting to remove N-linked glycosylations. Protein bands were visualized through V5. Do note that, because of the stronger denaturation conditions during the endo H treatment, V5 detection of the endo H treated samples was more efficient, giving thicker protein bands (although the same sample loading was used). PTK7 is a highly glycosylated protein (ten potential N-glycosylations) that is partially resistant to endo H treatment, resulting in only a minor fraction that is deglycosylated by endo H ( solid arrowhead ). CADA, cyclotriazadisulfonamide; Endo H, endoglycosidase H; HEK293T, human embryonic kidney 293T cells; huCD4, human CD4; PTK7, inactive tyrosine-protein kinase 7; SORT, sortilin.

Article Snippet: The endoplasmic reticulum lectin 1 (ERLEC1) expression vector (pGEM-T backbone) was purchased from Sinobiological, the PTK7 expression vector (pDONR223 backbone) from Addgene, and the DNAJ homolog subfamily C member 3 (DNAJC3) expression vector (pDONR223 backbone) from DNASU plasmid repository.

Techniques: Transfection, Labeling, Western Blot, Blocking Assay, Translocation Assay, SDS Page

Journal: Advanced Science

Article Title: POSTN‐Mediated Interplay of M1 Polarized Macrophage with Tendon‐Derived Stem Cells to Drive Traumatic Heterotopic Ossification Formation through PTK7/ATK Signaling?

doi: 10.1002/advs.202507951

Figure Lengend Snippet: POSTN enhances osteogenic propensity by binding to PTK7. A) Mass spectrometry analysis was used and presented through a Venn diagram to detect the potential molecules that showed increased binding to POSTN in the disease model between the sham group and the tendon injury group at 7 days, N = 3. B) Mass spectrum of the binding between PTK7 and POSTN. C) According to the Score Sequest HT, PTK7 ranks first among the molecules that bind to POSTN in the heterotopic ossification model. D) Docking images showing the predicted binding position of POSTN and PTK7 protein. E) IF staining showed co‐localization of POSTN and PTK7 in the cytoplasm of TDSCs, N = 3. F) IF staining showed co‐localization of POSTN and PTK7 in the heterotopic ossified tissue, N = 6. G) Co‐IP analysis was used to verified the bind relationship between POSTN and PTK7 in the osteogenic induced TDSCs, N = 3. H) Co‐IP analysis was used to verified the bind relationship between POSTN and PTK7 in the heterotopic ossified tissue, N = 3. I) Immunofluorescence staining indicated that overexpression of PTK7 could upregulate the protein expression levels of LCAD and MCAD in the absence of POSTN in vitro, N = 3. J) ALP and ARS staining was used to detect the osteogenesis of TDSCs in the POSTN‐/‐ and POSTN‐/‐&OV‐PTK7 groups, N = 6, **** p < 0.0001. K) Immunofluorescence staining for the Runx2 of tendons and micro‐CT analysis of HO formation in the POSTN‐/‐ and POSTN‐/‐&OV‐PTK7 groups, N = 6, **** p < 0.0001.

Article Snippet: The CD86 (Cat# 13395‐1‐AP), CD80 (Cat# 66406‐1‐lg), TLR2 (Cat# 66645‐1‐lg), Runx2 (Cat# 20700‐1‐AP), OPN (Cat# 22952‐1‐AP), OCN (Cat# 23418‐1‐AP), CD9 (Cat# 60232‐ 1‐lg), CD81 (Cat# 27855‐1‐AP), ALIX (Cat# 12422‐1‐AP), TSG101 (Cat# 28283‐1‐AP), Calnexin (Cat# 10427‐2‐AP), PTK7 (Cat# 17799‐1‐ AP), LCAD (Cat# 17526‐1‐AP), IL1β (Cat# 226048‐1‐AP), TNFα (Cat# 26162‐1‐AP), AMPK(Cat# 10929‐2‐AP), AKT(, Cat# 80457‐1‐RR) antibodies and p‐AKT(Ser473) (Cat# 66444‐1‐ Ig) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Binding Assay, Mass Spectrometry, Staining, Co-Immunoprecipitation Assay, Immunofluorescence, Over Expression, Expressing, In Vitro, Micro-CT

Journal: Advanced Science

Article Title: POSTN‐Mediated Interplay of M1 Polarized Macrophage with Tendon‐Derived Stem Cells to Drive Traumatic Heterotopic Ossification Formation through PTK7/ATK Signaling?

doi: 10.1002/advs.202507951

Figure Lengend Snippet: PTK7 mediates the osteogenic effect in TDSCs through AKT1. A) Phosphorylation sequencing analysis was used to detect the effects of PTK7 on downstream proteins., N = 3. B) The mass spectrometry diagram showing the increased phosphorylation at the S124 site of the AKT protein C) WB analysis was used to detect the expression of PTK7 could regulate the protein expression levels of p‐AKT in the TDSCs, N = 3, **** p < 0.0001. D) Immunofluorescence staining indicated that overexpression of PTK7 could upregulate the p‐AKT in the TDSCs, N = 3. E) Docking images showing the predicted binding position of PTK7 and AKT protein. F) Co‐IP analysis was used to verified the bind relationship between PTK7 and AKT in the osteogenic induced TDSCs, N = 3. G) Immunofluorescence staining indicated that overexpression of PTK7 could upregulate the p‐AKT in the TDSCs, N = 3. H) Immunofluorescence staining for LCAD and MCAD of TDSCs in the sh‐PTK7 and sh‐PTK7&OV‐S124 groups, N = 3. I) WB analysis was used to detect the expression of LCAD and MCAD proteins of TDSCs in the sh‐PTK7 and sh‐PTK7&OV‐S124 groups, N = 3, *** p < 0.001, **** p < 0.0001. J) ALP and ARS staining were used to detect the osteogenesis of TDSCs in the sh‐PTK7 and sh‐PTK7&OV‐S124 groups, N = 6, **** p < 0.0001.

Article Snippet: The CD86 (Cat# 13395‐1‐AP), CD80 (Cat# 66406‐1‐lg), TLR2 (Cat# 66645‐1‐lg), Runx2 (Cat# 20700‐1‐AP), OPN (Cat# 22952‐1‐AP), OCN (Cat# 23418‐1‐AP), CD9 (Cat# 60232‐ 1‐lg), CD81 (Cat# 27855‐1‐AP), ALIX (Cat# 12422‐1‐AP), TSG101 (Cat# 28283‐1‐AP), Calnexin (Cat# 10427‐2‐AP), PTK7 (Cat# 17799‐1‐ AP), LCAD (Cat# 17526‐1‐AP), IL1β (Cat# 226048‐1‐AP), TNFα (Cat# 26162‐1‐AP), AMPK(Cat# 10929‐2‐AP), AKT(, Cat# 80457‐1‐RR) antibodies and p‐AKT(Ser473) (Cat# 66444‐1‐ Ig) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Phospho-proteomics, Sequencing, Mass Spectrometry, Expressing, Immunofluorescence, Staining, Over Expression, Binding Assay, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: POSTN‐Mediated Interplay of M1 Polarized Macrophage with Tendon‐Derived Stem Cells to Drive Traumatic Heterotopic Ossification Formation through PTK7/ATK Signaling?

doi: 10.1002/advs.202507951

Figure Lengend Snippet: POSTN promotes the osteogenic transition of TDSCs by mediating the phosphorylation of AKT at the S124 site. A) High‐throughput sequencing was performed between TDSCs treated with SFs derived from M1 macrophages and SFs derived from M1 macrophages with the POSTN protein knocked out respectively and between overexpressed phosphorylation at the AKT S124 site and the mutated S124 site respectively. An intersection Venn diagram was drawn. B) WB analysis was used to detect the expression of CPT1 proteins of TDSCs in the M1‐SFs, POSTN‐/‐, sh‐PTK7, and mut‐S124 groups, N = 3, **** p < 0.0001. C) Immunofluorescence staining for LCAD and MCAD of TDSCs in the POSTN‐/‐, POSTN‐/‐&sh‐CPT1 and mut‐S124&sh‐CPT1 groups, N = 3. D) ALP and ARS staining were used to detect the osteogenesis of TDSCs in the POSTN‐/‐, POSTN‐/‐&sh‐CPT1 and mut‐S124&sh‐CPT1 groups, N = 6, **** p < 0.0001. E) WB analysis was used to detect the expression of LCAD and MCAD proteins of TDSCs in the POSTN‐/‐, POSTN‐/‐&sh‐CPT1 and mut‐S124&sh‐CPT1 groups, N = 3, **** p < 0.0001.

Article Snippet: The CD86 (Cat# 13395‐1‐AP), CD80 (Cat# 66406‐1‐lg), TLR2 (Cat# 66645‐1‐lg), Runx2 (Cat# 20700‐1‐AP), OPN (Cat# 22952‐1‐AP), OCN (Cat# 23418‐1‐AP), CD9 (Cat# 60232‐ 1‐lg), CD81 (Cat# 27855‐1‐AP), ALIX (Cat# 12422‐1‐AP), TSG101 (Cat# 28283‐1‐AP), Calnexin (Cat# 10427‐2‐AP), PTK7 (Cat# 17799‐1‐ AP), LCAD (Cat# 17526‐1‐AP), IL1β (Cat# 226048‐1‐AP), TNFα (Cat# 26162‐1‐AP), AMPK(Cat# 10929‐2‐AP), AKT(, Cat# 80457‐1‐RR) antibodies and p‐AKT(Ser473) (Cat# 66444‐1‐ Ig) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Phospho-proteomics, Next-Generation Sequencing, Derivative Assay, Expressing, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Representative western blot analysis of protein tyrosine kinase 7 (PTK7) expression in prostate tissues. The expression of PTK7 protein was significantly increased in prostate cancer tissues compared with benign prostatic hyperplasia tissues. Protein samples obtained from benign prostatic hyperplasia tissues and prostate cancer tissues were analysed using SDS-PAGE followed by immunoblotting with antibody against PTK7. The levels of β-actin were used as an internal control.

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Western Blot, Expressing, SDS Page, Control

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Immunohistochemical staining for PTK7 in prostate cancer and benign prostate tissue (original magnification ×200). ( A ) High PTK7 protein expression was found in cytoplasm of prostate cancer tissues; ( B ) Low PTK7 protein expression was found in cytoplasm of prostate cancer tissues; ( C ) PTK7 weakly positive staining was found in cytoplasm of benign prostate tissue; and ( D ) Negative immunostaining of negative controls with the primary antibody omitted in prostate cancer tissues.

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Clinicopathologic variables and PTK7 expression in 180 prostate cancer patients.

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Factors predictive of lymph node metastasis in the univariable analysis.

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Factors predictive of lymph node metastasis in the multivariable analysis.

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Associations between PTK7 expression and biochemical recurrence free time after radical prostatectomy in prostate cancer patients. Patients with high PTK7 expression showed significantly shorter biochemical recurrence free survival than those with low PTK7 expression ( p < 0.001, log-rank test).

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Prognostic value of PTK7 expression for the biochemical recurrence-free survival in univariate and multivariate analyses by Cox regression.

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Protein Tyrosine Kinase 7 (PTK7) as a Predictor of Lymph Node Metastases and a Novel Prognostic Biomarker in Patients with Prostate Cancer

doi: 10.3390/ijms150711665

Figure Lengend Snippet: Prognostic value of PTK7 expression for the overall survival in univariate and multivariate analyses by Cox regression.

Article Snippet: The filters were blocked at 4 °C overnight with blocking buffer (pH = 7.6) containing 5% nonfat dry milk and incubated with mouse anti-human monoclonal PTK7 antibody (MAB4499, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, which specifically recognizes the PTK7 protein.

Techniques: Expressing

Journal: Cell reports. Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma.

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: Figure 5. PTK7 CAR T cells have specific in vitro cytotoxicity (A) Twelve-hour flow cytometry cytotoxicity assay. Percentage cytotoxicity = the sum of 7AAD+, annexin V+, and 7AAD+ annexin V+ cells, gated on target cells only. n = 3 donors with n = 2 biological replicates. Four-hour cytotoxicity shown in Figure S5A. (B) Target cells and CAR T cells or mock T cell control were incubated with caspase-3/7 dye and live cell analysis was performed at the indicated time points. Processing definitions of caspase-3/7 activity (green object count) on target cells were determined. n = 1 donor. Error bars represent SD of 4 technical replicates. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Recombinant Human PTK7 Fc Chimera Protein R&D Systems 9799-TK-050 Recombinant Mouse PTK7 Fc Chimera Protein R&D Systems 10333-TK-050 Recombinant Human IL-2 PeproTech 200-02-50UG Recombinant Human IL-7 PeproTech 200-07-50UG CD3/CD28 Human T-Activator Dynabeads ThermoFisher Scientific 111.31D Polybrene EMD Millipore TR-1003 D-Luciferin PerkinElmer 122799 Accutase Stemcell technologies 7920 Biocytin hydrazide Biotium 90060 Sep-Pak tC18 vacuum cartridge Waters WAT054960 NeutrAvidin agarose resins Thermo Scientific 29201 TMT10plex Thermo Scientific Blasticidin CellGro 30-100-RB Puromycin Gemini 400-128P Doxorubicin Selleck Chemicals S1208 Melphalan Selleck Chemicals S8266 Topotecan Selleck Chemicals/McKesson Pharmaceuticals S9321/1109275 Critical commercial assays EasySep Human T cell Isolation Kit Stemcell technologies 17951 MycoAlert contamination kit Lonza LT07-318 Deposited data DepMap (DepMap, Broad (2022): DepMap 22Q2 Public. figshare.

Techniques: In Vitro, Cytometry, Cytotoxicity Assay, Control, Incubation, Cell Analysis, Activity Assay

Journal: Cell Death & Disease

Article Title: FOXP4-mediated induction of PTK7 activates the Wnt/β-catenin pathway and promotes ovarian cancer development

doi: 10.1038/s41419-024-06713-7

Figure Lengend Snippet: A The mRNA correlation between FOXP4 and PTK7 in OV from TCGA database. B The spearman correlation between the expression of FOXP4 and PTK7 in all tumor types in TCGA. Red represents positive expression and green represents negative expression. C Real-time PCR analysis of FOXP4 and PTK7 mRNA levels in A2780 cells transfected with EV or FOXP4 expression vector, n = 3. ** p < 0.01 vs EV, *** p < 0.001 vs EV. D Immunoblot analysis showing FOXP4 and PTK7 protein expression in A2780 cells transfected with EV or FOXP4. E Immunoblot analysis of FOXP4 in A2780 and OVCAR8 cells stably expressing either pLKO or shRNAs targeting FOXP4. F Schematic diam shows human PTK7 gene promoter and a putative FOXP4 binding site. TSS: transcription start site. G Schematic diam shows the human PTK7 WT and Mut promoters. The potential binding sites for FOXP4 were highlighted with yellow. H The activities of the human PTK7 WT and Mut promoters in 293T cells transfected with EV or FOXP4 was measured, n = 3. *** p < 0.001 vs EV. I Two shFOXP4s were co-transfected with the indicated plasmid into 293T cells for 36 h. The luciferase activity was then measured, n = 3. *** p < 0.001 vs pLKO. J , K Chip-PCR assay detecting FOXP4 binding to the human PTK7 promoter in A2780 cells expressing stable FOXP4, n = 3. *** p < 0.001 vs IgG. L A2780 cells were treated with ICG-001 or LiCl to detect PTK7 protein levels. M The luciferase activities of TCF/LEF1 in A2780 and OVCAR8 cells transfected with pLKO or shRNAs-PTK7 were measured, n = 3. ** p < 0.01 vs pLKO, *** p < 0.001 vs pLKO. N The luciferase activities of TCF/LEF1 in A2780 and OVCAR8 cells transfected with EV or PTK7 were measured, n = 3. ** p < 0.01 vs EV.

Article Snippet: The following primary antibodies were used: anti-β-catenin (Santa Cruz, sc-7963), anti-FOXP4 (Santa Cruz, sc-390892), anti-PTK7 (Bethyl Laboratories, A304-451A), anti-Cyclin D1 (Abcam, ab134175), anti-GSK3B (Abcam, ab76225), anti-c-Myc (Abcam, ab32072), anti-E-cadherin (Abcam, ab40772), anti-N-cadherin (Abcam, AB207608), anti-Vimentin (Abcam, ab92547), and anti-β-actin (Santa Cruz, sc-8432).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Stable Transfection, Binding Assay, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: FOXP4-mediated induction of PTK7 activates the Wnt/β-catenin pathway and promotes ovarian cancer development

doi: 10.1038/s41419-024-06713-7

Figure Lengend Snippet: A PTK7 was retrovirally infected into A2780 cells stably expressing FOXP4-shRNA. Lysates of A2780 cells with pLKO, sh-FOXP4, and sh-FOXP4 + PTK7 were immunoblotted with the indicated antibodies. B The cell growth curve of cells from ( A ), n = 3. ** p < 0.01. C Incorporation of BrdU in cells from ( A ) was measured by ELISA, n = 3. ** p < 0.01, *** p < 0.001. D Clonogenic assay of cells in ( A ), n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. E Relative migration rates of cells in ( A ) in a transwell assay, n = 3. ** p < 0.01, *** p < 0.001. F A2780 cells stably expressing pLKO or sh-FOXP4 and stably expressing sh-FOXP4 + PTK7 were inoculated subcutaneously in nude mice, respectively, and the folded line graphs indicate the average volume of tumors over time, n = 6. *** p < 0.001. G Image representing the tumor on day 28, n = 6. H Bar graphs represent the weight of the tumor on day 28, n = 6. *** p < 0.001.

Article Snippet: The following primary antibodies were used: anti-β-catenin (Santa Cruz, sc-7963), anti-FOXP4 (Santa Cruz, sc-390892), anti-PTK7 (Bethyl Laboratories, A304-451A), anti-Cyclin D1 (Abcam, ab134175), anti-GSK3B (Abcam, ab76225), anti-c-Myc (Abcam, ab32072), anti-E-cadherin (Abcam, ab40772), anti-N-cadherin (Abcam, AB207608), anti-Vimentin (Abcam, ab92547), and anti-β-actin (Santa Cruz, sc-8432).

Techniques: Infection, Stable Transfection, Expressing, shRNA, Enzyme-linked Immunosorbent Assay, Clonogenic Assay, Migration, Transwell Assay

Journal: Cell Death & Disease

Article Title: FOXP4-mediated induction of PTK7 activates the Wnt/β-catenin pathway and promotes ovarian cancer development

doi: 10.1038/s41419-024-06713-7

Figure Lengend Snippet: A The FOXP4 retrovirus was infected into A2780 cells stably expressing sh-PTK7, and A2780 cell lysates of pLKO, FOXP4, and FOXP4+sh-PTK7 were immunobloted with the indicated antibodies. B The cell growth curve of cells from ( A ), n = 3. *** p < 0.001. C Incorporation of BrdU in cells from ( A ) was measured by ELISA, n = 3. * p < 0.05, ** p < 0.01. D Clonogenic assay of cells in ( A ), n = 3. *** p < 0.001. E Relative invasion rates of cells in ( A ) in a transwell assay, n = 3. *** p < 0.001. F A2780 cells stably expressing EV or FOXP4 or FOXP4 infected with sh-PTK7 in FOXP4 were inoculated subcutaneously in nude mice, respectively, and the folded line graphs indicate the average volume of tumors over time, n = 6. *** p < 0.001. G Image representing the tumor on day 28, n = 6. H Bar graphs represent the weight of the tumor on day 28, n = 6. *** p < 0.001.

Article Snippet: The following primary antibodies were used: anti-β-catenin (Santa Cruz, sc-7963), anti-FOXP4 (Santa Cruz, sc-390892), anti-PTK7 (Bethyl Laboratories, A304-451A), anti-Cyclin D1 (Abcam, ab134175), anti-GSK3B (Abcam, ab76225), anti-c-Myc (Abcam, ab32072), anti-E-cadherin (Abcam, ab40772), anti-N-cadherin (Abcam, AB207608), anti-Vimentin (Abcam, ab92547), and anti-β-actin (Santa Cruz, sc-8432).

Techniques: Infection, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Clonogenic Assay, Transwell Assay

Journal: Cell Death & Disease

Article Title: FOXP4-mediated induction of PTK7 activates the Wnt/β-catenin pathway and promotes ovarian cancer development

doi: 10.1038/s41419-024-06713-7

Figure Lengend Snippet: In this working model, we depict a signaling pathway involving Wnt/β-catenin/TCF4-FOXP4-PTK7. FOXP4 acts as a transcriptional target of Wnt/β-catenin/TCF4 signaling and enhances the transcription of PTK7, consequently promoting the development and progression of OV tumors.

Article Snippet: The following primary antibodies were used: anti-β-catenin (Santa Cruz, sc-7963), anti-FOXP4 (Santa Cruz, sc-390892), anti-PTK7 (Bethyl Laboratories, A304-451A), anti-Cyclin D1 (Abcam, ab134175), anti-GSK3B (Abcam, ab76225), anti-c-Myc (Abcam, ab32072), anti-E-cadherin (Abcam, ab40772), anti-N-cadherin (Abcam, AB207608), anti-Vimentin (Abcam, ab92547), and anti-β-actin (Santa Cruz, sc-8432).

Techniques: