Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: CBX6 expression is downregulated in multiple cancer types (A) Statistical analysis of The Cancer Genome Atlas (TCGA) Pan-Cancer RNA sequencing (RNA-seq) database shows CBX6 expression is downregulated in various cancer types. (B) CBX6 expression is significantly suppressed in brain cancer, breast cancer, lung adenocarcinoma (abbreviated as LUAD), and prostate cancer, based on analysis of TCGA tumor microarray data using the BRowse All Variants Online (BRAVO) database method. (C and D) Comparison of CBX6 expression in patient-derived GBM tissues and primary tumor cells relative to respective normal controls using qRT-PCR. GAPDH was used as a loading control. (D) Reduced CBX6 expression is significantly associated with poor prognosis in patients with glioma, as shown in Kaplan-Meier survival curves derived from a public patient-derived microarray database, p = 1.94∗ e−7. PBT, primary brain tumor; RBT, recurrent brain tumor.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Expressing, RNA Sequencing, Microarray, Comparison, Derivative Assay, Quantitative RT-PCR, Control

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: CBX6 modulation affects tumor cell morphology and proliferation (A) CBX6 modulation affects the morphology of U-251 MG cells in two-dimensional culture. (B) Downregulation of CBX6 increases growth and invasion of U-251 MG cells in 3D culture. (C) Overexpression of CBX6 significantly suppresses U-251 MG proliferation on days 3 and 5 post-plating, as measured by MTT assay. (D) MTT assay of KLuc cells stably overexpressing human CBX6 or transfected with control plasmid on days 3 and 5 post-plating. (E) Silencing CBX6 increases U-251 MG proliferation compared to shRNA controls. Images were captured randomly from different fields under 20× magnification. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” indicated p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Over Expression, MTT Assay, Stable Transfection, Transfection, Control, Plasmid Preparation, shRNA, Knockdown

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: Effects of CBX6 modulation on invasion and migration of U-251 MG cells (A and B) Representative images and cell quantification from transwell migration assays of U-251 MG cells with modulated CBX6 expression compared to corresponding controls. (C and D) Representative images and cell quantification from transwell invasion assays of U-251 MG cells with modulated CBX6 expression compared to respective controls. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” Indicates p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Migration, Expressing, Plasmid Preparation, Control, Over Expression, Knockdown

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: Effect of human CBX6 overexpression on glioma tumor growth (A and B) Bioluminescent imaging of NSG mice (A) and quantification of bioluminescence signal intensity as fold change (B) on days 4 and 8 ( n = 5). (C) Bioluminescent imaging at week 2 showing reduced Kluc tumor size in mice with human CBX6 overexpression compared to controls ( n = 10). (D and E) Kaplan-Meier survival analysis demonstrating that overexpression of human CBX6 in U-251 MG cells (D) ( n = 5) and Kluc cells (E) ( n = 10) improved mouse survival. (F) Histological analysis of tumor invasion and microsatellite metastasis in CBX6-overexpressing tumors. “∗” Indicates p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Over Expression, Imaging

Journal: Molecular Therapy Oncology

Article Title: CBX6 and CA9 as predictive indicators and therapeutic targets in GBM

doi: 10.1016/j.omton.2026.201159

Figure Lengend Snippet: CBX6 binds to the CA9 promoter (A) Genes related to tumor cell invasion, proliferation, or migration were selected based on RNA sequencing data from U-251 MG cells with CBX6 knockdown compared to negative controls. (B) qRT-PCR results show that CA9 expression is affected by CBX6 dysregulation in U-251 MG cells, with overexpression or shRNA-mediated knockdown of CBX6, using GAPDH as a reference gene. (C) Gene expression correlation analysis from CGGA reveals an inverse relationship between CBX6 and CA9 expression patterns ( http://www.cgga.org.cn ). (D) Western blot and qRT-PCR data demonstrate changes in CBX6 and CA9 expression in U-251 MG and PBT030 cells under normoxic (N) and hypoxic (H) conditions for 24 and 48 h, using 28S as a reference gene. (E) ChIP assay results from CBX6-overexpressing U-251 MG cells show detection of the CA9 promoter sequence using two primer sets in CBX6/Flag pull-down products compared to a negative IgG control via qRT-PCR. VC, vector control; OE, CBX6 overexpression; KD, CBX6 knockdown. “∗” Indicates p < 0.05.

Article Snippet: A pCMV6-entry plasmid containing Myc-DDK (same as Flag)-tagged human CBX6 cDNA (Cat#: RC204166) and a CBX6 Human shRNA Plasmid Kit (Cat#: TF314170) were purchased from OriGene.

Techniques: Migration, RNA Sequencing, Knockdown, Quantitative RT-PCR, Expressing, Over Expression, shRNA, Gene Expression, Western Blot, Sequencing, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Experimental protocol for intervention of SuHx-PH in SOX17enhKO mice shows administration of the RUNX1 inhibitor Ro5-3335 or Ro24-7429 every other day for 6 times 1 week after the beginning of mild SuHx treatment. (B and C) RVSP (B) and RV/LV+S ratio (C) were measured at the end of week 3. Data in (B) and (C) are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques:

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Representative Western blot analyses of endothelial and hematopoietic markers in control versus RUNX1 overexpression HPAECs, with quantification of relative band intensities normalized to loading controls. (B and C) Human Endothelial Cell Biology RT²-PCR Array analyses in HPAECs overexpressing RUNX1 compared to control vector transduced cells. Gene expression values were normalized to housekeeping genes and represented as fold change relative to controls. Bar graphs and Volcano plots highlight differentially expressed genes meeting the significance criteria of fold change > 1.5 and FDR < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Western Blot, Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Gene Expression

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: HPAECs transduced with RUNX1 overexpressing (RUNX1 OE) or control lentivirus were subjected to quantitative assays to evaluate RUNX1-driven phenotypic and functional changes. (A) Morphometric assessment (length/width ratio) demonstrated pronounced alterations in cell morphology in RUNX1 OE cells. (B) Tube formation assays demonstrated impaired angiogenic network assembly (C) Migration assays showed increased gap closure over time in RUNX1 OE cells. (D) MTS proliferation assays indicated enhanced growth in RUNX1 OE cells. (E) Reduced Caspase-3/7 activity following TNF-α treatment demonstrated decreased apoptosis in RUNX1 OE cells. All image-based analyses (A-C) were quantified using ImageJ software. Statistical significance for (D and E) was determined by two-way ANOVA with multiple comparisons, while all other comparisons were performed using unpaired two-tailed t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Transduction, Control, Functional Assay, Migration, Activity Assay, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Western blot analyses show (A) increased RUNX1 protein levels in SOX17 KO HPAECs, with quantification of relative band intensity normalized to β-ACTIN. (B and C) Treatment of SOX17 KO HPAECs with RUNX1 inhibitor Ro5-3335 at 0, 10 or 25 µM (B) or RUNX1-targeting siRNA (C) leads to partial restoration of endothelial marker expression in the absence of SOX17. *p < 0.05.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Western Blot, Marker, Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A and B) RT-PCR analyses (A) and Western blot analyses (B) demonstrate increased RUNX1 expression and reduced endothelial marker levels in iPSC-ECs derived from SOX17 mutant patients compared with healthy controls. (C) Western blots shows in a dose-dependent manner RUNX1 inhibitor Ro5-3335 partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. (D) Western blots shows RUNX1 targeting siRNA downregulation partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 denote statistical significance.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Marker, Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: RUNX1 expression in whole bone marrow (A) and whole lung (B) in SOX17enhKO and WT mice after 3 weeks of normoxia (Norm) or Sugen/hypoxia (SuHx). N = 5-15 mice per group, * P < 0.05, ** P < 0.01, ns: not significant.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic SRV mice were subjected to mild SuHx treatment. When the SRV mice were treated with corn oil without tamoxifen, they developed significantly elevated RVSP. When the SRV mice were treated with tamoxifen and placed under mild SuHx conditions, they exhibited normal RVSP. **** P < 0.0001.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Out of the 359 PAH patients, SOX17 expression was undetectable in 206 and detectable in 153 patients, whereas the expression of RUNX1 could be seen in all subjects. RUNX1 expression was significantly higher in patients with undetectable expression of SOX17 compared to those with detectable SOX17 expression. * P < 0.05.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Machine learning-based biomarker selection: ( A ) LASSO regression curve for feature selection in the training cohort GSE71220 . The x-axis shows the log of the regularization parameter λ, and the y-axis shows the partial likelihood deviance. Two vertical dashed lines indicate the optimal λ values: the left dashed line corresponds to λ.min (the value that minimizes the cross-validated error), and the right dashed line corresponds to λ.1se (the largest λ within one standard error of the minimum). The optimal penalty parameter λ = 0.013 (λ.min) was selected, yielding 21 non-zero coefficient genes including HK3 , GPR15 , and RORC . ( B ) LASSO coefficient paths. Each colored line represents the trajectory of a gene coefficient as the regularization penalty increases (decreasing log λ). The vertical dashed line indicates the selected λ.min at log λ = −4.3182, corresponding to the optimal model complexity. ( C ) Boruta feature importance assessment. Boxplots display the importance scores of each feature across iterative random forest runs. The y-axis represents the importance score; the vertical position (median) of each boxplot reflects the feature’s importance, while the box length represents the interquartile range, indicating variability across runs. Core predictors (green) are distinguished from tentative (blue) and rejected (red) features. ( D ) Venn diagram showing the overlap between genes selected by LASSO and Boruta. Six hub genes ( GPR15 , HK3 , RORC , CLEC4D , TCF7 , and SLC4A10 ) were identified by both algorithms and retained for further analysis. ( E ) Validation of biomarker expression in the training and validation cohorts. Violin plots display the expression levels of the six hub genes. The upper panels show expression in the training cohort GSE71220 , and the lower panels show expression in the internal validation cohort GSE42057 significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Biomarker Discovery, Selection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Validation of the diagnostic performance of the three-gene signature: ( A ) Receiver operating characteristic curves for the three biomarkers in the training cohort GSE71220 . The area under the curve values are 0.777 for RORC , 0.771 for CLEC4D , and 0.752 for TCF7 . RORC is shown as a green stepped line, CLEC4D in red, and TCF7 in blue. ( B ) Receiver operating characteristic curves in the internal validation cohort GSE42057 . The area under the curve values are 0.662 for RORC , 0.644 for CLEC4D , and 0.680 for TCF7 . Color coding follows the same scheme as in panel A. ( C ) Nomogram for predicting COPD probability based on the expression levels of the three-gene signature. Each gene contributes a score corresponding to its expression value; the total score is summed to estimate the predicted probability. As an example, a randomly selected patient with a total score of 156 corresponds to a predicted probability of 0.983. ( D ) Decision curve analysis evaluating the clinical utility of the three-gene signature. The curves represent the net benefit of clinical decisions based on RORC , TCF7 , and CLEC4D , and the combined nomogram across a range of threshold probabilities.

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Biomarker Discovery, Diagnostic Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: External validation of the three-gene signature across independent cohorts: ( A ) Validation in the peripheral blood cohort GSE56766 . The violin plots show the expression levels of the three biomarkers in patients with COPD and healthy controls. CLEC4D was significantly upregulated, while RORC and TCF7 did not show statistically significant differences. The receiver operating characteristic curves for each biomarker are shown to the right, with the area under the curve values ranging from 0.602 to 0.654. RORC is depicted in blue, CLEC4D in orange, and TCF7 in green. ( B ) Validation in the peripheral blood cohort GSE306950 . The violin plots show that RORC and TCF7 were significantly downregulated, whereas CLEC4D was significantly upregulated in the COPD samples compared to controls. The corresponding ROC curves yielded area under the curve values of 0.976 for RORC , 0.776 for CLEC4D , and 0.776 for TCF7 . Color coding follows the same scheme as in panel ( A ). ( C ) Validation in the lung tissue cohort GSE106986 . The violin plots show that both RORC and TCF7 were significantly downregulated in COPD lung tissue, while CLEC4D did not reach statistical significance. The receiver operating characteristic analysis revealed area under the curve values of 0.886 for RORC , 0.571 for CLEC4D , and 0.814 for TCF7 . Color coding is consistent with previous panels. Significance: * p < 0.05, *** p < 0.001.

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Biomarker Discovery, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Gene set enrichment analysis and regulatory network construction: ( A ) Gene set enrichment analysis of the three biomarkers. The GSEA enrichment plots display the top five significantly enriched pathways for RORC , CLEC4D , and TCF7 . Positive enrichment for RORC in neuroactive ligand–receptor interaction (NES = 3.05) and negative enrichment in spliceosome and ubiquitin−mediated proteolysis pathways (NES < −2.7) are shown. For CLEC4D , positive enrichment in spliceosome, ubiquitin−mediated proteolysis, and proteasome pathways, and negative enrichment in neuroactive ligand–receptor interaction and olfactory transduction are displayed. For TCF7 , positive enrichment in the ribosome and primary immunodeficiency pathways (NES = 2.14) and negative enrichment in the complement and coagulation cascades are presented. ( B ) Transcription factor−miRNA−mRNA regulatory network for TCF7 and RORC . The network was constructed using the GSE24709 dataset and visualized with Cytoscape. The nodes represent transcription factors (triangles), miRNAs (diamonds), and target genes (circles). IRF4 , FOXM1 , and EGR1 are shown as transcription factors connecting TCF7 and RORC . The network is presented in a blue color theme. ( C ) Gene–gene interaction network for TCF7 , CLEC4D , and RORC . Interactions were retrieved from GeneMANIA, integrating evidence from multiple public databases and published studies. Edge colors represent seven interaction types: physical interactions (77.64%), co−expression (8.01%), predicted interactions (5.37%), co−localization (3.63%), genetic interactions (2.87%), pathway (1.88%), and shared protein domains (0.60%). ( D ) Transcription factor−miRNA−mRNA regulatory network highlighting miRNA connections. hsa−miR−485−5p is indicated as a key miRNA connecting TCF7 and RORC . The network follows the same blue color theme as panel ( B ).

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Ubiquitin Proteomics, Olfactory, Transduction, Coagulation, Construct, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Immune landscape and biomarker associations: ( A ) Heatmap of ssGSEA-based quantification for 28 immune cell types in patients with COPD versus healthy controls. Red indicates higher relative abundance, and blue indicates lower abundance. ( B ) Correlation heatmap of differentially abundant immune cell subpopulations. Spearman correlations are represented by color intensity (red: positive; blue: negative). ( C ) Heatmap of immune−related functional pathway enrichment scores in COPD versus controls. ( D – F ) Correlation heatmaps illustrating the associations between the three diagnostic biomarkers ( CLEC4D , RORC , and TCF7 ) and ( D ) immune cell subpopulations, ( E ) functional pathways, and ( F ) HLA gene expression levels. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical significance is denoted as follows: ****, p < 0.0001.

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Biomarker Discovery, Functional Assay, Diagnostic Assay, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Single-cell transcriptomic landscape and intercellular communication analysis: ( A ) UMAP visualization of the GSE249584 single-cell RNA-seq dataset. The cells were clustered into eight distinct immune cell types based on marker gene expression. ( B ) UMAP plots showing the expression distribution of CLEC4D , RORC , and TCF7 in the control and COPD samples. CLEC4D was predominantly expressed in myeloid populations, whereas RORC and TCF7 were largely restricted to lymphoid lineages. ( C ) Violin plots comparing the expression levels of the three biomarkers between control and COPD groups across different cell types. CLEC4D showed upregulation in monocytes, macrophages, and dendritic cells in the COPD samples. RORC expression was downregulated in T cells and natural killer cells, whereas TCF7 exhibited elevated expression in natural killer and B cell clusters in COPD compared to controls. * p < 0.05, ** p < 0.01. ( D ) CellChat network analysis showing the overall communication strength and weight among immune cell populations in control and COPD conditions. Edge thickness represents the probability of intercellular communication. ( E ) Bubble plot displaying significant ligand–receptor pairs between cell populations in control and COPD groups. Circle size indicates communication probability, and color intensity represents relative expression levels.

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Single Cell, RNA Sequencing, Marker, Gene Expression, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: qRT−PCR validation of the three−gene signature in CSE−stimulated (COPD−like) cellular models. Expression levels of CLEC4D , RORC , and TCF7 were measured by quantitative real−time PCR in THP−1−derived macrophages (for CLEC4D ) and Jurkat T cells (for RORC and TCF7 ) following 24 h of stimulation with cigarette smoke extract: ( A ) CLEC4D expression was significantly upregulated in CSE−stimulated macrophages compared to control ( p < 0.001). In contrast, ( B , C ) RORC and TCF7 expression were significantly downregulated in CSE−stimulated Jurkat T cells compared to control ( p < 0.001 for both). Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using a two−tailed Student’s t -test. Significance: *** p < 0.001.

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: Histological and immunofluorescence validation of TCF7 in human lung tissue: ( A ) H&E staining and separate immunofluorescence channels for DAPI (blue), CD8 + T cells (green), and TCF7 (red) in healthy lung tissues. ( B ) Merged immunofluorescence image of healthy lung tissue. Scale bar is 100 μm. ( C ) Magnified merged immunofluorescence image of healthy lung tissue. Scale bar is 25 μm. ( D ) H&E staining and separate immunofluorescence channels for DAPI (blue), CD8 + T cells (green), and TCF7 (red) in COPD lung tissues. ( E ) Merged immunofluorescence image of COPD lung tissue. Scale bar is 100 μm. ( F ) Magnified merged immunofluorescence image of COPD lung tissue. Scale bar is 25 μm.

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Immunofluorescence, Biomarker Discovery, Staining

Journal: International Journal of Molecular Sciences

Article Title: Unfolding Immune Dysregulation in COPD: Identification of a Three-Gene Signature and Functional Validation of TCF7 in Human Lung Tissue and T Lymphocytes

doi: 10.3390/ijms27104231

Figure Lengend Snippet: TCF7 regulates pro−caspase−8 expression in T lymphocytes and is significantly reduced in COPD. ( A ) Immunofluorescence co−staining of control human lung tissue displaying separate channels for DAPI (blue), caspase−8 (green), TCF7 (red), and the merged image. Scale bar is 50 μm. ( B ) Immunofluorescence co−staining of COPD human lung tissue displaying separate channels for DAPI (blue), caspase−8 (green), TCF7 (red), and the merged image. Note the marked reduction in both TCF7 and caspase−8 signals compared to the control. Scale bar is 50 μm. ( C ) Representative Western blot images of TCF7 (50 kDa), pro−caspase−8 (55 kDa), and internal control β−tubulin (55 kDa) in wild type (WT) and TCF7 knockout (KO) Jurkat T cells. ( D ) Quantitative densitometric analysis of TCF7 protein levels comparing WT and KO groups. ( E ) Quantitative densitometric analysis of pro−caspase−8 protein levels comparing WT and KO groups. ( F ) Representative Western blot images of TCF7 and β−tubulin in primary T lymphocytes isolated from the peripheral blood of healthy donors (Control) and patients with COPD (Model). ( G ) Quantitative densitometric analysis of TCF7 protein levels in human primary T lymphocytes. ( H ) Representative Western blot images of TCF7 and β−tubulin protein levels in Jurkat T cells across four experimental conditions including Control, shRNA, shRNA plus TCF7 Rescue construct, and shRNA plus Empty Vector. ( I ) Quantitative densitometric analysis of TCF7 protein levels across the four experimental rescue groups. ( J ) Representative Western blot images of pro−caspase−8 and β−tubulin protein levels across the same four experimental conditions in Jurkat T cells. ( K ) Quantitative densitometric analysis of pro−caspase−8 protein levels across the four experimental rescue groups. Data in the bar charts are presented as mean ± SD ( n = 4 for primary human cells, n = 3 for cell line experiments). Statistical significance was assessed using Student’s t test with Welch’s correction where appropriate (* p < 0.05, *** p < 0.001, ns indicates not significant).

Article Snippet: For rescue experiments, a full-length human TCF7 cDNA clone (OriGene, Cat. No. RC224338) was utilized.

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, Knock-Out, Isolation, shRNA, Construct, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Chimeric Anti-Glypican 1 Antibodies Exert Antitumor Activities in Xenograft Models of Lung and Pancreatic Cancers

doi: 10.3390/ijms27104181

Figure Lengend Snippet: Production of recombinant G 1 Mab-28-mG 2a and G 1 Mab-28-hG 1 . ( A ) After determination of CDRs of G 1 Mab-28 (mouse IgG 1 ), G 1 Mab-28-mG 2a (mouse IgG 2a ) and G 1 Mab-28-hG 1 (human IgG 1 ) were produced. The amino acid sequence of V H and V L CDRs was indicated. ( B ) PMab-231 (control mIgG 2a ), G 1 Mab-28-mG 2a , humCvMab-62 (control hIgG 1 ), and G 1 Mab-28-hG 1 were subjected to SDS-PAGE, and the gel was stained with Bio-Safe CBB G-250 Stain. ( C ) Flow cytometry using G 1 Mab-28-mG 2a (1 μg/mL; Red line) and G 1 Mab-28-hG 1 (1 μg/mL; Blue line) against CHO/GPC1 and CHO/PA16-mouse GPC1 (CHO/mouse GPC1). An anti-PA16 tag mAb (NZ-1) detected PA16-tagged mouse GPC1 (1 μg/mL; Green line). After treatment with primary mAbs or buffer control (Black line), cells were treated with Alexa Fluor 488-conjugated anti-mouse or rat IgG, or FITC-conjugated anti-human IgG. ( D ) Determination of the binding affinity of G 1 Mab-28-mG 2a and G 1 Mab-28-hG 1 . CHO/GPC1 was suspended in G 1 Mab-28-mG 2a and G 1 Mab-28-hG 1 at indicated concentrations, followed by Alexa Fluor 488-conjugated anti-mouse IgG or FITC-conjugated anti-human IgG treatment. The SA3800 Cell Analyzer was used to analyze fluorescence data. The dissociation constant ( K D ) values were determined using GraphPad Prism 6.

Article Snippet: The mouse GPC1 ( NM_016696.4 ) cDNA was obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Recombinant, Produced, Sequencing, Control, SDS Page, Staining, Flow Cytometry, Binding Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Chimeric Anti-Glypican 1 Antibodies Exert Antitumor Activities in Xenograft Models of Lung and Pancreatic Cancers

doi: 10.3390/ijms27104181

Figure Lengend Snippet: ADCC and CDC by G 1 Mab-28-mG 2a against GPC1-positive tumor cells. The target cells labeled with Calcein AM (CHO/GPC1, PC-10, and PK-45H) were incubated with effector splenocyte derived from BALB/c nude mice ( A ) or rabbit complement ( B ) in the presence of G 1 Mab-28-mG 2a or control mIgG 2a . Calcein release into the medium was measured, and cytotoxicity was determined. Values are shown as the mean ± SEM. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01; two-tailed unpaired t -test).

Article Snippet: The mouse GPC1 ( NM_016696.4 ) cDNA was obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Labeling, Incubation, Derivative Assay, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Chimeric Anti-Glypican 1 Antibodies Exert Antitumor Activities in Xenograft Models of Lung and Pancreatic Cancers

doi: 10.3390/ijms27104181

Figure Lengend Snippet: Antitumor activity of G 1 Mab-28-mG 2a against human tumor xenografts. ( A – C ) CHO/GPC1 ( A ), PC-10 ( B ), and PK-45H ( C ) cells were subcutaneously injected into BALB/c nude mice (day 0). G 1 Mab-28-mG 2a (100 μg) or control mIgG 2a (100 μg) were intraperitoneally injected into each mouse on days 7 and 13 (arrows). The tumor volume is represented as the mean ± SEM. ** p < 0.01 (two-way ANOVA with Sidak’s post hoc test). ( D – F ) After cell inoculation, the mice were euthanized on day 20. The tumor weights (left) and appearance (right) of CHO/GPC1 ( D ), PC-10 ( E ), and PK-45H ( F ) xenografts were measured. Values are presented as the mean ± SEM. ** p < 0.01 (two-tailed unpaired t -test). Scale bar, 1 cm. ( G – I ) Body weight (mean ± SEM) of xenograft-bearing mice treated with mAbs is presented. There is no significant difference (two-way ANOVA with Sidak’s post hoc test).

Article Snippet: The mouse GPC1 ( NM_016696.4 ) cDNA was obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Injection, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Chimeric Anti-Glypican 1 Antibodies Exert Antitumor Activities in Xenograft Models of Lung and Pancreatic Cancers

doi: 10.3390/ijms27104181

Figure Lengend Snippet: ADCC and CDC by G 1 Mab-28-hG 1 against GPC1-positive tumor cells. The target cells labeled with Calcein AM (CHO/GPC1, PC-10, and PK-45H) were incubated with effector splenocyte derived from BALB/c nude mice ( A ) or rabbit complement ( B ) in the presence of G 1 Mab-28-hG 1 or control hIgG 1 . Calcein release into the medium was measured, and cytotoxicity was determined. Values are shown as the mean ± SEM. Asterisks indicate statistical significance (* p < 0.05; two-tailed unpaired t -test).

Article Snippet: The mouse GPC1 ( NM_016696.4 ) cDNA was obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Labeling, Incubation, Derivative Assay, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Chimeric Anti-Glypican 1 Antibodies Exert Antitumor Activities in Xenograft Models of Lung and Pancreatic Cancers

doi: 10.3390/ijms27104181

Figure Lengend Snippet: Antitumor activity of G 1 Mab-28-hG 1 against human tumor xenografts. ( A – C ) CHO/GPC1 ( A ), PC-10 ( B ), and PK-45H ( C ) cells were subcutaneously injected into BALB/c nude mice (day 0). G 1 Mab-28-hG 1 (100 μg) or control hIgG 1 (100 μg) were intraperitoneally injected into each mouse on days 7 and 13 (arrows). The tumor volume is represented as the mean ± SEM. * p < 0.05, ** p < 0.01 (two-way ANOVA with Sidak’s post hoc test). ( D – F ) After cell inoculation, the mice were euthanized on day 20. The tumor weights (left) and appearance (right) of CHO/GPC1 ( D ), PC-10 ( E ), and PK-45H ( F ) xenografts were measured. Values are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 (two-tailed unpaired t -test). Scale bar, 1 cm. ( G – I ) Body weight (mean ± SEM) of xenograft-bearing mice treated with the mAbs is presented. There is no significant difference (two-way ANOVA with Sidak’s post hoc test).

Article Snippet: The mouse GPC1 ( NM_016696.4 ) cDNA was obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Injection, Control, Two Tailed Test