Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Single-cell communication networks. (A) Incoming communication patterns of target cells, showing pathways to which each cell type responds. (B) Outgoing communication patterns of secreting cells, illustrating the pathways through which cells send signals, MIF, MK and CXCL pathway exhibit high activity. (C) Network diagram showing the strength of intercellular communication, with connections between various cell types. (D) Scatter plot comparing outgoing and incoming communication strengths across cell populations, with bubble size indicating the number of interactions, malignant cells have higher strength of intercellular communication. (E) Chord diagram depicting communication via the MK pathway between different cell types. (F) Ligand-receptor interaction probabilities within the MK pathway between malignant and other cell types. Dot size represents significance (P-value), and color represents communication probability highlighting the MDK-NCL signaling pathway. (G) Violin plots of MK pathway gene expression levels across cell types, showing gene activity variations, MDK has advancer expression level in malignant cells.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Activity Assay, Gene Expression, Expressing

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Spatial transcriptomics and MDK-NCL signal communication. (A) Niche clustering in spatial transcriptomics samples, identifying distinct ecological zones. (B) Spatial expression of representative markers in key regions: MUC1 (tumor region), LYZ (immune region), COL14A1 (stromal region), and SFTPC (normal region). (C) Violin plots displaying the expression of MUC1, LYZ, COL14A1, and SFTPC across different niches. (D) MCPcounter analysis showing the infiltration of six cell types (e.g., endothelial cells, fibroblasts, immune lineages) across spatial regions. (E) Spatial niche classification, distinguishing tumor, immune-stromal, and normal regions. (F) MDK-NCL ligand-receptor interaction analysis, spatially mapping MDK ligands, NCL receptors, and their binding regions.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Single-cell pseudotime analysis. (A) Pseudotime trajectory analysis showing the 6 differentiation states of cells. (B) Subtype classification of malignant cells along the pseudotime trajectory. (C) Pseudotime scores mapped along the differentiation trajectory. (D) UMAP plot visualizing pseudotime scores across individual cells. (E) Box plots comparing pseudotime scores across different malignant cell clusters, cluster 0, 1, and 5 had higher pseudotime scores. (F) UMAP plot of differentiation states, with colors representing distinct states. (G) Stacked bar plots showing the proportion of differentiation states within each malignant cell cluster, cluster 0, 1, and 5 have larger proportion of state 6. (H) Expression dynamics of MK pathway genes (e.g., MDK, NCL, ITG genes) along the pseudotime trajectory, highlighting gene expression changes during differentiation, MDK and NCL express more in the later time.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Association of MDK-NCL with the immune microenvironment. (A) Boxplot shows the expression levels of MDK and NCL genes in tumor and control groups, it exhibit higher activity in tumor group. (B) MDK-NCL enrichment scores in tumor and control groups. (C) Relative mRNA expression levels of MDK and NCL in tumor and control groups from in-house data. (D) Relative protein expression levels of MDK and NCL in tumor and control groups from in-house data. (E) Comparison of MDK protein expression levels between tumor and control groups. (F) Comparison of NCL protein expression levels between tumor and control groups. (G) Correlation of MDK and NCL expression with ImmuneScore, StromalScore, ESTIMATEScore, and TumorPurity. (H) Scatter plots depicting the relationship between MDK and NCL expression and immune-related scores (ImmuneScore, StromalScore, ESTIMATEScore) as well as TumorPurity. (I) Comparison of immune cell infiltration scores across high and low MDK-NCL expression groups for 28 immune cell types. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Control, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Association of MDK-NCL with immunotherapy response. (A) Comparison of tumor mutation burden (TMB) between high and low MDK-NCL expression groups. (B) Comparison of microsatellite instability (MSI) between high and low MDK-NCL groups. (C) Comparison of dysfunction scores between high and low MDK-NCL groups. (D) Comparison of exclusion scores between high and low MDK-NCL groups. (E) Expression of immunogenic cell death (ICD)-related genes in high and low MDK-NCL groups. (F) Expression levels of CTLA4 and PD1 in high and low MDK-NCL groups. (G) Comparison of immune checkpoint gene expression between high and low MDK-NCL expression groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Comparison, Mutagenesis, Expressing, Gene Expression

Journal: Disease Models & Mechanisms

Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

doi: 10.1242/dmm.022350

Figure Lengend Snippet: Expression of P3 suppressed nucleolar stress in cells expressed with expanded-CAG RNA. (A) Amino acid sequence of nucleolin (NCL) peptides used in this study. (B) The P3 and P5 peptides disrupted the interaction between expanded-CAG RNA and NCL. After in vitro binding of CAG 78 RNA and GST-NCL protein in the presence of NCL peptides, reverse-transcription PCR was performed to detect the binding of CAG 78 RNA to GST-NCL. (C) Amino acid sequences of mutant (MT) P3 peptides. The mutated residues are underlined. (D) Expression of P3WT resumed the expression level of pre-45s rRNA in EGFP CAG78 RNA-expressing HEK293 cells. Real-time PCR was performed to determine the expression level of pre-45s rRNA in cells co-transfected with EGFP CAG and P3 constructs. (E) Expression of P3WT resumed the physical interaction between NCL and upstream control element (UCE) in EGFP CAG78 RNA-expressing HEK293 cells. Chromatin immunoprecipitation was performed. Real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. (F) Expression of P3WT resumed the DNA methylation status of UCE. ‘–’ represents cells that were transfected with pcDNA3.1 empty vector. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. The enzyme-treated DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. (G) Expression of P3WT suppressed caspase 9 activity in HEK293 cells expressing EGFP CAG78 RNA. Experiments were repeated at least three times and data are expressed as mean±s.d. *** P <0.001.

Article Snippet: Purified nucleolin protein (GST-NCL) was purchased from Abnova (Taiwan), and the control GST protein was expressed and purified as mentioned in .

Techniques: Expressing, Sequencing, In Vitro, Binding Assay, Reverse Transcription, Mutagenesis, Real-time Polymerase Chain Reaction, Transfection, Construct, Control, Chromatin Immunoprecipitation, DNA Methylation Assay, Plasmid Preparation, Methylation, Amplification, Activity Assay