Journal: Frontiers in Immunology

Article Title: Identification of immune-related prognostic biomarkers in lung squamous cell carcinoma microenvironment

doi: 10.3389/fimmu.2025.1724319

Figure Lengend Snippet: Flowchart of the study illustrating the construction and analysis of the immune-related gene (IRG) prognostic signature for LUSC.

Article Snippet: The human LUSC cell lines Calu-1 and NCI-H520, and the human monocytic cell line THP-1 were obtained from the American Type Culture Collection (ATCC).

Techniques:

Journal: Frontiers in Immunology

Article Title: Identification of immune-related prognostic biomarkers in lung squamous cell carcinoma microenvironment

doi: 10.3389/fimmu.2025.1724319

Figure Lengend Snippet: Results of immune cell infiltration between high- and low-risk groups. (A, B) Histogram of immune cell infiltration. (C) Box plot of differential expression of 22 immune cells. (D) Proportion of 22 types of immune cells in LUSC patients. (E) Comparison of stromal cells in high-risk and low-risk groups. (F–L) The correlation between characteristic genes and immune cells. Significance levels indicated are based on Benjamini-Hochberg adjusted P -values (FDR). (*p < 0.05).

Article Snippet: The human LUSC cell lines Calu-1 and NCI-H520, and the human monocytic cell line THP-1 were obtained from the American Type Culture Collection (ATCC).

Techniques: Quantitative Proteomics, Comparison

Journal: Frontiers in Immunology

Article Title: Identification of immune-related prognostic biomarkers in lung squamous cell carcinoma microenvironment

doi: 10.3389/fimmu.2025.1724319

Figure Lengend Snippet: NR1D2 is downregulated in LUSC, inhibits tumor cell migration, invasion, and modulates the immune microenvironment. (A–D) Immunohistochemistry (A, C) and Western blot (B, D) analysis show significantly reduced NR1D2 expression in LUSC tissues compared to adjacent normal lung tissues (P < 0.001). Representative images (A, B) and quantification (C, D) are shown. (E–G) Efficient knockdown of NR1D2 in Calu1 and NCI-H520 cells using two independent siRNAs (si NR1D2 #1 and si NR1D2 #2) confirmed by Western blot (E, F) and qPCR (G) (P <0.001 vs. siCtrl). (H, I) Wound healing assays demonstrate significantly impaired migratory capacity in NR1D2 -silenced Calu1 and NCI-H520 cells. Representative images (H) at 0h and 24h and quantification of wound closure (I) are shown (P < 0.001 vs. siCtrl). (J–L) Transwell assays showing significantly reduced migration and invasion of Calu1 and NCI-H520 cell lines following NR1D2 silencing. Representative images (J) and quantification of migrated/invaded cells (K, L) are presented (P < 0.001 vs. siCtrl). (M–O) Flow cytometry analysis of THP-1-derived macrophages cultured in conditioned medium (CM) from NR1D2 -silenced tumor cells. Enhanced polarization towards CD206+CD163+ M2-like macrophages was observed. Representative dot plots (M) and quantification of CD206 + CD163 + macrophage following exposure to CM from NR1D2 -depleted Calu1 cells (N) and NCI-H520 cells (O) subsets are shown (**P < 0.001 vs. CM from siCtrl cells). (P, Q) ELISA assays show increased secretion of IL-10 (P) and TGF-β1 (Q) in macrophage supernatants following exposure to CM from NR1D2 -depleted tumor cells (*P < 0.05, **P < 0.01, ***P <0.001 vs. CM from siCtrl cells). All data are presented as mean ± SD. Scale bars are provided in the respective image panels.

Article Snippet: The human LUSC cell lines Calu-1 and NCI-H520, and the human monocytic cell line THP-1 were obtained from the American Type Culture Collection (ATCC).

Techniques: Migration, Immunohistochemistry, Western Blot, Expressing, Knockdown, Flow Cytometry, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay