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: Journal of Neuroinflammation

Article Title: Cholecystokinin ameliorates cognitive impairment via inhibiting microglia phagocytosis of excitatory synapses in sepsis-associated encephalopathy mice

doi: 10.1186/s12974-025-03554-9

Figure Lengend Snippet: Dorsal hippocampal CA1 region CCK8 injection ameliorates cognitive dysfunction in SAE model mice. (A) Cannula implantation site and experimental timeline. The percentages of freezing time in the (B) training phase, (C) context test, and (D) tone test were recorded on day 3 after LPS injection. (E) Swimming speed and (F) escape latency were recorded in the training phase. (G) Swimming speed, (H) latency to the platform, (I) number of platform crossings, and (J) time spent in the target quadrant were recorded in the MWM spatial probe test on day 4 after LPS injection. (K) Swimming trajectories. Data are expressed as the mean ± SD ( n = 11–12 per group). Data B-D and G-J were analysed by two-way ANOVA followed by Bonferroni post hoc test; data E and F were analysed by repeated measures ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group; # p < 0.05 vs. LPS group

Article Snippet: Next, to select the effective dose of CCK8 for inhibiting C1q expression, different doses of CCK8 (0.2, 0.5, 1, and 2 μM, Tocris, Bristol, UK, Cat. No. 1166) were used.

Techniques: Injection, Control

Journal: Journal of Neuroinflammation

Article Title: Cholecystokinin ameliorates cognitive impairment via inhibiting microglia phagocytosis of excitatory synapses in sepsis-associated encephalopathy mice

doi: 10.1186/s12974-025-03554-9

Figure Lengend Snippet: CCK8 ameliorates excitatory synaptic plasticity in the dorsal hippocampal CA1 region of SAE model mice. (A) Experimental timeline. (B, C) Western blot was used to detect the hippocampal protein levels of PSD95 and vGlut1 in mice. (D, E) Immunofluorescence was used to detect the numbers of excitatory synapses (white arrows) in the dorsal hippocampal CA1 region. (F) Representative traces for sEPSCs. Statistical results of (G) sEPSC frequency and (H) amplitude. (I) Representative traces for mEPSCs. Statistical results of (J) mEPSC frequency and (K) amplitude. Data are expressed as the mean ± SD ( n = 4–5 per group). All data were analysed by two-way ANOVA followed by Bonferroni post hoc test. ** p < 0.01, **** p < 0.0001 vs. control group; # p < 0.05 vs. LPS group

Article Snippet: Next, to select the effective dose of CCK8 for inhibiting C1q expression, different doses of CCK8 (0.2, 0.5, 1, and 2 μM, Tocris, Bristol, UK, Cat. No. 1166) were used.

Techniques: Western Blot, Immunofluorescence, Control

Journal: Journal of Neuroinflammation

Article Title: Cholecystokinin ameliorates cognitive impairment via inhibiting microglia phagocytosis of excitatory synapses in sepsis-associated encephalopathy mice

doi: 10.1186/s12974-025-03554-9

Figure Lengend Snippet: CCK8 inhibits C1q-mediated microglial phagocytosis of synapses and A1 astrocyte polarisation in SAE model mice. (A) Experimental timeline. (B) Venn diagram and heatmap showing differentially expressed genes in the control and LPS + CCK8 groups compared with the LPS group. (C) mRNA and (D, E) protein levels of C1q were detected by qPCR and western blot, respectively. Immunofluorescence was used to detect the number of PSD95 + C1q + with microglia (F, J), Iba1 fluorescence intensity (G), soma volume (H), branch length (I) and the colocalization of (K, L) GFAP with C3 in the dorsal hippocampal CA1 region of mice. (M, N) Western blot was used to detect GFAP and C3 protein levels in the hippocampus of mice. Data are expressed as the mean ± SD ( n = 4–5 per group). All data were analysed by two-way ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. LPS group

Article Snippet: Next, to select the effective dose of CCK8 for inhibiting C1q expression, different doses of CCK8 (0.2, 0.5, 1, and 2 μM, Tocris, Bristol, UK, Cat. No. 1166) were used.

Techniques: Control, Western Blot, Immunofluorescence, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Cholecystokinin ameliorates cognitive impairment via inhibiting microglia phagocytosis of excitatory synapses in sepsis-associated encephalopathy mice

doi: 10.1186/s12974-025-03554-9

Figure Lengend Snippet: CCK8 inhibits C1q expression via CCK2R in BV2 microglia. (A) Experimental timeline. (B–G, I–L) Western blot was used to detect C1q protein levels in the medium and in cells. (H) A Cell Counting Kit-8 was used to detect cell viability. Data are expressed as the mean ± SD ( n = 3 per group). Data B-E and H were analysed by one-way ANOVA followed by Bonferroni post hoc test, the remaining data were analysed by two-way ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. LPS group; ++ p < 0.01, +++ p < 0.001 vs. LPS + CCK8 group

Article Snippet: Next, to select the effective dose of CCK8 for inhibiting C1q expression, different doses of CCK8 (0.2, 0.5, 1, and 2 μM, Tocris, Bristol, UK, Cat. No. 1166) were used.

Techniques: Expressing, Western Blot, Cell Counting, Control