Journal: Oncology Reports

Article Title: NF-κB-driven LUZP1 promotes metastasis and chemoresistance in head and neck squamous cell carcinoma

doi: 10.3892/or.2026.9115

Figure Lengend Snippet: NF-κB signaling activation promotes LUZP1 expression in HNSCC cells. (A) The expression of LUZP1 in FaDu, OECM-1 and SAS cells treated with SB431542 (10 µM), LY294002 (10 µM), Rapamycin (10 µM), BAY 11–7085 (5 µM) or YC-1 (30 µM) was determined by western blot assay. Signal quantification was measured using ImageJ 1.54 g software (National Institutes of Health) and the relative intensity was normalized to untreated control. The red dashed line represents the normalized value as 1. (B) The expression of LUZP1 in FaDu, OECM-1 and SAS cells with or without BAY 11–7085 was validated by western blot assay. (C) Spearman's monotonic correlation between LUZP1 and NFKB1 or NFKB2 expression in HNSCC was analyzed using The Cancer Genome Atlas RNA-Sequencing database on the GEPIA server. (D) Protein expression of NF-κB p65 and LUZP1 in OECM-1 and SAS cells with NF-κB p65 knockdown (+) or control shRNA -), as determined by western blot analysis. β-actin, loading control. (E) IC 50 values of docetaxel in OECM-1 and SAS cells with or without NF-κB p65 knockdown. (F) IC 50 values of cisplatin in OECM-1 and SAS cells with or without NF-κB p65 knockdown. The expression of LUZP1 in different HNSCC cells treated with (G) IL-1β (3 ng/ml) or (H) TNFα (10 ng/ml) for the indicated times was determined by western blot assay. β-actin, loading control. (I) Transwell cell migration assay was conducted using OECM-1 cells with or without LUZP1 knockdown in the presence or absence of TNFα (10 ng/ml) treatment. Signal quantification using crystal violet extract was measured by colorimetric analysis at 570 nm, and the relative signal intensities were normalized to untreated shControl (shLUZP1, -; TNFα, -) (n=3). (J) Genomic visualization of the human LUZP1 locus (GRCh38/hg38) showing RefSeq-curated exon annotations, NF-κB RelA ChIP-seq binding signals in FaDu cells (ReMap), layered H3K27ac ChIP-seq profiles from ENCODE cell lines, and GeneHancer regulatory element annotations. Red boxes denote promoter regions and gray boxes indicate putative enhancers. Blue vertical bars mark the locations of ChIP-qPCR primer sets designed for experimental validation. (K) ChIP-qPCR analysis showing increased NF-κB (RelA) occupancy at the LUZP1 promoter in response to TNF-α treatment. For statistical analyses, (E and F) a 2-tailed unpaired Student's t -test; (I) a factorial two-way ANOVA, followed by Tukey's Honestly Significant Difference post hoc test. **P<0.01. TPM, transcripts per million; ChIP-seq, chromatin immunoprecipitation sequencing; LUZP1, leucine zipper protein 1; sh, short hairpin.

Article Snippet: The pharmacological inhibitors used in the present study included SB431542 (5 and 10 μM; cat. no. 616464; Merck KGaA), LY294002 (5 and 10 μM; cat. no. HY-10108; MedChemExpress), rapamycin (5 and 10 μM; cat. no. R0395; Merck KGaA), BAY 11-7085 (5 and 10 μM; cat. no. HY-10257; MedChemExpress), and YC-1 (10 and 30 μM; cat. no. sc-202856; Santa Cruz Biotechnology, Inc.).

Techniques: Activation Assay, Expressing, Western Blot, Software, Control, RNA Sequencing, Knockdown, shRNA, Cell Migration Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, Biomarker Discovery