Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: PLS3 is aberrantly expressed in HNSCC and associated with prognosis. ( A ) TIMER2.0 was utilized to detect the expression levels of PLS3 in different tumors within the TCGA database. ( B , C ) The expression level of PLS3 was analyzed in normal tissues and paired adjacent tumor tissues, including unmatched tissues ( B ) and matched tissues ( C ). ( D ) Relative PLS3 mRNA expression between normal tissue and HNSCC and cell lines. ( E ) Representative images of PLS3 expression in normal tissues and HNSCC tissues via IHC staining. Scale bar: 100 μm. ( F ) PLS3 IHC score assessment in normal and HNSCC tissues. ( G ) Survival analysis of patients with low vs. high PLS3 expression using Kaplan–Meier method. HR = hazard ratio. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as means ± SEM. Statistical analysis was performed via two-tailed Student’s t -test.

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: PLS3 knockdown impairs the proliferation and colony formation of HNSCC cells. ( A ) Relative PLS3 mRNA expression in HSC3 and SCC25 cells transfected with siPLS3 #1, siPLS3 #2, siPLS3 #3, or siNC (negative control siRNA), detected via qRT-PCR. ( B ) Western blot analysis of PLS3 protein expression in HSC3 and SCC25 cells after PLS3 knockdown. ( C ) CCK-8 assay evaluating cell proliferation of HSC3 and SCC25 cells with PLS3 knockdown at 0, 24, 48, 72, 96, and 120 h. ( D ) Colony formation images of HSC3 and SCC25 cells following PLS3 knockdown. ( E ) Statistical analysis of colony numbers in HSC3 and SCC25 cells with PLS3 knockdown. * p < 0.05, ** p < 0.01, *** p < 0.001. ns: no significance. Data are shown as means ± SEM, with statistical analysis performed via two-tailed Student’s t -test or ANOVA.

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Two Tailed Test

Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: PLS3 knockdown inhibits migration, invasion, and epithelial–mesenchymal transition (EMT) of HNSCC cells. ( A ) Wound healing assay images of HSC3 and SCC25 cells at 0 h and 24 h after PLS3 knockdown (siPLS3 #1, siPLS3 #2, siPLS3 #3) or siNC treatment. ( B ) Statistical analysis of wound healing closure rates in HSC3 and SCC25 cells, showing reduced migration ability after PLS3 knockdown. ( C ) Transwell invasion assay images of HSC3 and SCC25 cells with PLS3 knockdown, indicating decreased invasive capacity. ( D ) Quantitative analysis of invaded cell numbers in HSC3 and SCC25 cells, verifying the inhibitory effect of PLS3 knockdown on invasion. ( E ) Western blot analysis of PLS3, E-cadherin, N-cadherin, and Vimentin expression in HSC3 and SCC1 cells after shPLS3 or shNC treatment. ( F ) Quantitative densitometric analysis of Western blot bands for EMT-related proteins in shNC and shPLS3 groups. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as means ± SEM, with statistical analysis via two-tailed Student’s t -test or ANOVA.

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Knockdown, Migration, Wound Healing Assay, Transwell Invasion Assay, Western Blot, Expressing, Two Tailed Test

Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: PLS3 knockdown inhibits tumor growth in the in vivo xenograft model. ( A ) Representative images of tumors from shNC (control) and shPLS3 (PLS3 knockdown) groups. ( B ) Tumor volume growth curves over time, comparing shNC and shPLS3 groups. ( C ) Statistical analysis of final tumor volumes, showing suppressed growth in the shPLS3 group. ( D ) HE staining and immunohistochemical staining for PLS3 and Ki67 in tumor tissues of shNC and shPLS3 groups, with scale bars indicating magnification. ( E ) Quantitative analysis of PLS3 IHC scores and Ki67-positive cell rates, revealing reduced PLS3 expression and proliferation (Ki67) in the shPLS3 group. ** p < 0.01, *** p < 0.001. Data are presented as means, with statistical analysis performed via two-tailed Student’s t -test.

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Knockdown, In Vivo, Control, Staining, Immunohistochemical staining, Expressing, Two Tailed Test

Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: Functional enrichment analysis and immune correlation of PLS3 in HNSCC. ( A ) Scatter plot displaying the association results of PLS3, presenting Pearson correlation coefficient and −log10( p -value) via Pearson test. ( B ) Venn diagram showing the overlap between PLS3 co-expression genes and HNSCC survival-related genes. ( C ) GO (Gene Ontology) enrichment analysis of PLS3-associated genes, covering biological process, cellular component, and molecular function. ( D ) KEGG pathway enrichment analysis of PLS3-related genes, highlighting significantly enriched pathways. ( E ) Comparison of ESTIMATE scores (ESTIMATE Score, Immune Score, Stromal Score) between PLS3 low/high expression groups. In ( C , D ), p -adjust values reflect significance, with color gradients indicating different p -adjust ranges. ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Functional Assay, Expressing, Comparison

Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: Immune cell infiltration analysis and cell interaction related to PLS3 in HNSCC. ( A ) Immune cell infiltration correlation analysis via CIBERSORT and MCP-counter, and red font indicates statistically significant values ( p < 0.05). ( B ) UMAP (Uniform Manifold Approximation and Projection) visualization of cell types. ( C ) UMAP plot showing PLS3 expression distribution across cell clusters. ( D ) Proportion of cell types in high vs. low PLS3 expression groups, highlighting compositional differences.

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Expressing

Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: PLS3 knockdown inhibits tumor growth and enhances CD8 + T cell infiltration and cytotoxicity in xenograft models. ( A ) Representative images of tumors isolated from shNC and shPLS3 groups on day 12. ( B ) Quantitative analysis of tumor volume in shNC vs. shPLS3 groups (mean ± SD; * p < 0.05). ( C , D ) Flow cytometry plots showing the proportion of CD8 + T and CD4 + T cells in tumor-infiltrating lymphocytes (TILs) of shNC ( C ) and shPLS3 ( D ) groups. ( E ) Statistical comparison of CD8 + T cell proportion in TILs between shNC and shPLS3 groups (* p < 0.05). ( F , G ) Flow cytometry plots showing GZMB + expression in CD8 + T cells of shNC ( F ) and shPLS3 ( G ) groups. ( H ) Statistical analysis of GZMB + CD8 + T cell proportion in TILs between shNC and shPLS3 groups (* p < 0.05). ( I , J ) Flow cytometry plots showing GZMB + expression in CD4 + T cells of shNC ( I ) and shPLS3 ( J ) groups. ( K ) Statistical comparison of GZMB + CD4 + T cell proportion in TILs between shNC and shPLS3 groups (ns: no significance).

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Knockdown, Isolation, Flow Cytometry, Comparison, Expressing

Journal: Cancers

Article Title: PLS3 Is a Prognostic Biomarker and Correlates with Immune Infiltrates in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers17233882

Figure Lengend Snippet: Correlation of PLS3 expression with immune characteristics. ( A ) Heatmap showing the correlation between PLS3 and immunoinhibitors across multiple tumor types, with HNSCC highlighted by a red box. ( B ) Scatter plots and Spearman correlation analysis of PLS3 expression with specific immunoinhibitors (LGALS9, ADORA2A, PDCD1, CD96) in 522 HNSCC samples. ( C ) Heatmap illustrating the correlation between PLS3 and chemokines, with HNSCC marked by a red box. ( D ) Scatter plots and Spearman correlation analysis of PLS3 expression with chemokines (XCL2, CCL19, CX3CL1, CXCL13) in HNSCC. ( E ) Heatmap depicting the correlation between PLS3 and chemokine receptors, with HNSCC highlighted via a red box. ( F ) Scatter plots and Spearman correlation analysis of PLS3 expression with chemokine receptors (CXCR4, CXCR3, CXCR5, CCR7) in HNSCC. In ( B , D , F ), rho represents the Spearman correlation coefficient, and p -value indicates statistical significance. The heatmaps ( A , C , E ) use color gradients (red to blue) to reflect positive to negative correlation strengths.

Article Snippet: Sections were blocked with 10% goat serum (Boster Biological Technology, Wuhan, China; Cat. AR0009) for 30 min at room temperature to block nonspecific binding, then incubated overnight at 4 °C with primary anti-PLS3 antibody (Proteintech, cat. 12917-1-AP, 1:400 dilution).

Techniques: Expressing

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Primers for RT‐PCR analysis and ChIP assay

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Sequencing

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Reporter gene assay with constructs containing the pivotal region of the p21 promoter. (A) Schematic of four reporter gene constructs encompassing nucleotides −2308 to +204 of the p21 promoter, with sequences identical except for rs3829963 (–2119C/A) and rs2395655 (–809G/A) polymorphic sites. Luciferase expression in the four constructs in ESCC cells EC ‐109 (B) and KYSE 150 (C) were standardized by cotransfection with pRL ‐ SV 40. LEDGF /p75 expression vector was cotransfected to evaluate its potential effect on the activity of the p21 promoter, using pc DNA 3.0 empty vector as control. Fold increase was measured by defining the activity of the pGL 3‐Basic vector and pc DNA 3.0 empty vector as 1. The value of each reported construct was calculated as the mean fold increase ± SD from three independent experiments each performed in duplicate (top panel). LEDGF /p75 (p75 in brief) protein levels in EC ‐109 and KYSE 150 cells were examined by Western blot analysis (bottom panel). * P < 0.05 and ** P < 0.01 vs rs2395655 A allele‐containing counterparts, respectively

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Reporter Gene Assay, Construct, Luciferase, Expressing, Cotransfection, Plasmid Preparation, Activity Assay, Western Blot

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: LEDGF /p75 interacted with human genomic DNA sequence containing rs2395655 G allele. (A) The rs2395655 A>G transition created one cis‐regulatory element, that is STRE element ( AGGGGT , nucleotides −810 to −805), in the p21 promoter. Polymorphic sites of rs2395655 were italicized. (B) Nuclear extracts from KYSE 150 cells were used for electrophoretic mobility shift assay ( EMSA ) with two biotin‐labeled probes carrying rs2395655 G and A allele, respectively. Anti‐ LEDGF /p75 antibody (C‐16, 2 mg), IgG control antibody or a 400‐fold molar excess of cold probe was added to the binding reaction to examine the specificity of the protein‐ DNA complex formation. The black and hollow arrows indicated the shift and supershift bands, respectively. (C) Chromatin immunoprecipitation was performed using anti‐ LEDGF /p75 antibody (C‐16) and genomic DNA mixture of EC ‐109 and KYSE 410 cells, carrying rs2395655 GG and AA genotype, respectively. Human p21 region of interest was amplified and sequenced. Human GAPDH and FBXO 10 loci were included as respective negative and positive LEDGF /p75 binding controls

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Sequencing, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Chromatin Immunoprecipitation, Amplification

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: LEDGF /p75 regulated the expression of p21 gene in ESCC cells with rs2395655 GG genotype. (A) Specific small interference RNA s (si‐p75‐1 and si‐p75‐2)‐mediated depletion of LEDGF /p75 (p75 in brief) induced obviously down‐regulated expression of p21 mRNA in EC ‐109 and KYSE 150 cells carrying rs2395655 GG genotype but not KYSE 410 and KYSE 450 cells with rs2395655 AA genotype. (B) Ectopic expression of p75 apparently increased the expression level of p21 mRNA in EC ‐109 and KYSE 150 cells, while no change of the p21 expression level was detected in KYSE 410 and KYSE 450 cells. For specific si RNA transfection and ectopic expression of p75, the mismatched si RNA (mock) and empty vector transfections were used as control, respectively. The relative signal intensity of each target gene was quantified and normalized to internal control, β‐actin. The graph indicates the relative mRNA expression levels of p75 and p21 and the values are mean ± SD for three independent experiments. M, DNA marker. Neg, negative control. * P < 0.01 and ** P > 0.05 vs controls, respectively

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing, Transfection, Plasmid Preparation, Marker, Negative Control

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Immunohistochemistry staining of the p21 protein in ESCC tissues. No‐anti p21, negative control with primary antibody replaced by PBS . Anti‐p21, the brown signals represented positive staining for p21 protein and the staining was scored on a scale as indicated in the Materials and Methods. Positive cases were defined as those with moderate to strong p21 nuclear staining in ≥ 10% of tumor cells. Magnification, × 100 (top panel) and × 400 (bottom panel), respectively

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Immunohistochemistry, Staining, Negative Control

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Associations of clinical characteristics and genetic factors with the p21 protein expression of ESCC patients (n = 218)

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing, Cell Differentiation, Variant Assay

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Kaplan–Meier survival estimation of 218 ESCC patients related to possible predictors. (A) Elevated p21 protein expression indicated better postoperative outcome for ESCC patients (32.0 and 19.0 months of median survival for p21‐positive and negative patients, respectively, P = 0.001). (B) The median survival time of ESCC patients with rs2395655 GG genotype was significantly increased compared with rs2395655 AA or AG genotype (30.0 vs 20.0 months, P = 0.003)

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing

Journal: Cancer Medicine

Article Title: Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma

doi: 10.1002/cam4.2067

Figure Lengend Snippet: Independent predictors of disease‐free survival time in multivariate analysis in ESCC patients (n = 218)

Article Snippet: After preincubating in 10% goat serum for nonspecific binding block, slides were then incubated overnight at 4°C with mouse anti‐human p21 protein (BD Biosciences; 1:500 dilution).

Techniques: Expressing, Variant Assay