Journal: Molecular Medicine Reports

Article Title: Liquid-liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression

doi: 10.3892/mmr.2025.13720

Figure Lengend Snippet: LLPS phenomenon of PHLDB2 in OSCC. (A) Prediction of disordered protein regions (IUPred2) and disordered binding motifs (ANCHOR2) in PHLDB2 via the IUPred2A platform. (B) LLPS propensity scoring of PHLDB2 using PhaSePred. In vitro droplet assay images of Alexa Fluor 488-labeled PHLDB2 condensates under increasing (C) protein or (D) NaCl concentrations (scale bar, 10 µm). (E) EGFP-PHLDB2 plasmid-transfected SCC1 cells showing intracellular droplet formation (scale bar, 10 µm). (F) FRAP-based quantification of normalized fluorescence intensity in EGFP-PHLDB2 condensates within live OSCC cells. (G) FRAP recovery time-lapse imaging of EGFP-PHLDB2 droplets in live OSCC cells (scale bar, 10 µm). FRAP, fluorescence recovery after photobleaching; LLPS, liquid-liquid phase separation; OSCC, oral squamous cell carcinoma.

Article Snippet: PHLDB2 protein solutions (cat. no. Ag27331; Proteintech Group, Inc.) was conjugated with Alexa Fluor 488 (cat. no. ab236553; Abcam).

Techniques: Binding Assay, In Vitro, Labeling, Plasmid Preparation, Transfection, Fluorescence, Imaging

Journal: Molecular Medicine Reports

Article Title: Liquid-liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression

doi: 10.3892/mmr.2025.13720

Figure Lengend Snippet: Promoting effects of PHLDB2 on proliferation, stemness, invasion and metastasis in OSCC cell lines. (A) PHLDB2 mRNA levels were decreased in OSCC cells transfected with PHLDB2-targeting siRNAs (si1/si2). ***P<0.001, ****P<0.0001 vs. control-si. (B) Western blotting confirmed PHLDB2 protein knockdown following siRNA transfection. OD values demonstrated changes in (C) HSC3 cells and (D) SCC1 cells upon PHLDB2 depletion. ***P<0.001. Tumor spheres formed in (E) HSC3 cells and (F) SCC1 cells. Scale bar, 100 µm. (G) Sphere number decreased in PHLDB2-knockdown HSC3/SCC1 cells. ***P<0.001 vs. control-si. (H) Sphere diameter decreased in PHLDB2-knockdown HSC3/SCC1 cells. **P<0.01 vs. control-si. Sphere-forming capacity decreased in (I) HSC3 cells and (J) SCC1 cells with PHLDB2 knockdown. (K) Representative image of colony formation assays showed reduction in PHLDB2-knockdown HSC3/SCC1 cells vs. control. Scale bar, 3 mm. (L) Quantification of colony formation assays showed reduction in PHLDB2-knockdown HSC3/SCC1 cells. ***P<0.001, ****P<0.0001 vs. control-si. (M) Representative image of invasion assays indicated suppression in PHLDB2-knockdown HSC3/SCC1 cells. Scale bar, 50 µm. (N) Quantification of invasion assays indicated suppression in PHLDB2-knockdown HSC3/SCC1 cells. ***P<0.001 vs. control-si. (O) Representative image of wound healing assays indicated depletion of migratory capacity in PHLDB2-knockdown HSC3 cells vs. control. Scale bar, 100 µm. (P) Wound healing assays indicated depletion of migratory capacity in PHLDB2-knockdown HSC3 cells. ***P<0.001 vs. control-si. (Q) Quantification of wound healing assays indicated depletion of migratory capacity in PHLDB2-knockdown SCC1 cells vs. control. Scale bar, 100 µm. (R) Wound healing assays indicated depletion of migratory capacity in PHLDB2-knockdown SCC1 cells. *P<0.05, **P<0.01 vs. control-si. OSCC, oral squamous cell carcinoma; si, small interfering.

Article Snippet: PHLDB2 protein solutions (cat. no. Ag27331; Proteintech Group, Inc.) was conjugated with Alexa Fluor 488 (cat. no. ab236553; Abcam).

Techniques: Transfection, Control, Western Blot, Knockdown

Journal: Molecular Medicine Reports

Article Title: Liquid-liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression

doi: 10.3892/mmr.2025.13720

Figure Lengend Snippet: Exploration of the molecular mechanisms of PHLDB2 promoting the malignant characteristics of OSCC. (A) Differential gene analysis identified differentially expressed genes between PHLDB2 low- and high-expression groups in TCGA-OSCC cohort. Threshold: P<0.05 and |log2FC|>1. (B) Differential gene analysis identified differentially expressed genes between the PHLDB2 knockdown group and the control group. Thresholds: P<0.05 and |log2FC|>1. (C) Intersection analysis revealed downregulated pathways shared between PHLDB2-RNAseq and PHLDB2-TCGA enrichment profiles. (D) Integrated Hallmark pathway analysis demonstrated enriched pathway for downregulated genes. (E) Integrated GO pathway analysis identified enriched biological terms for downregulated genes. (F) Integrated KEGG pathway analysis showed enriched pathways for downregulated genes. (G) Heatmap displayed expression patterns of PI3K-Akt signaling pathway genes in PHLDB2-knockdown vs. control groups. Reverse transcription-quantitative PCR indicated reduced PIK3CA expression in PHLDB2-depleted (H) HSC3 and (I) SCC1 cells. **P<0.01, ***P<0.001 vs. control-si. GO, Gene Ontology; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; OSCC, oral squamous cell carcinoma; RNAseq, RNA sequencing; TCGA, The Cancer Genome Atlas; MsigDB, Molecular Signatures Database; si, small interfering.

Article Snippet: PHLDB2 protein solutions (cat. no. Ag27331; Proteintech Group, Inc.) was conjugated with Alexa Fluor 488 (cat. no. ab236553; Abcam).

Techniques: Expressing, Knockdown, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, RNA Sequencing

Journal: Molecular Medicine Reports

Article Title: Liquid-liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression

doi: 10.3892/mmr.2025.13720

Figure Lengend Snippet: High expression of PHLDB2 is associated with a poorer prognosis for patients with OSCC. (A) Expression levels of PHLDB2 in tumor and normal tissues from TCGA-OSCC cohort. (B) PHLDB2 expression in paired tumor and normal samples from TCGA-OSCC cohort. (C) PHLDB2 levels in OSCC tissues and normal mucosa in the GSE213862 cohort. (D) PHLDB2 expression in early-stage (T1/T2) and late-stage (T3/T4) tumors from TCGA-OSCC. (E) Survival comparison between the PHLDB2 high and low expression groups in TCGA-OSCC. (F and G) IHC staining of PHLDB2 in tumor and matched normal tissues (Sun Yat-Sen University cohort; scale bar, 50 µm). Semi-quantitative IHC analysis of (H) tumor vs. normal tissues; (I) LN + vs. LN − tissues; and (J) G1 vs. G2/G3 tissues. *P<0.05, ***P<0.001 and ****P<0.0001. CI, confidence interval; IHC, immunohistochemical; LN, lymph node metastasis; OSCC, oral squamous cell carcinoma; TCGA, The Cancer Genome Atlas.

Article Snippet: PHLDB2 protein solutions (cat. no. Ag27331; Proteintech Group, Inc.) was conjugated with Alexa Fluor 488 (cat. no. ab236553; Abcam).

Techniques: Expressing, Comparison, Immunohistochemistry, Immunohistochemical staining