Journal:

Article Title: Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner

doi:

Figure Lengend Snippet: ILK-induced AP-1 activity is mediated by c-jun. (A) In this band shift assay, serum-exposed HEK-293 cells were transfected by Lipofectin with 0.5 μg of each of the indicated cDNAs. PD98059 (25 μM) was added to the medium 12 h prior to harvesting the cells. Nuclear extracts (2 μg) from the transfected cells were incubated with 32P-end-labeled AP-1 consensus oligonucleotide containing the protein binding site. Reaction products were analyzed on a nondenaturing 5% polyacrylamide gel (left gel). The specificity of complex formation was established by a competition experiment using cold AP-1 oligonucleotide as the competitor (right gel). For immunoblot studies, 10 μg of protein was resolved by SDS–10% PAGE. c-jun (Ser-73) phosphorylation, c-jun protein expression level, ERK phosphorylation (Tyr-204), and ERK protein expression level (not shown) were determined by Western blot analysis. ILK and GSK-3 expression levels in the transfected cells were evaluated by Western blot analysis using anti-V5 and anti-HA antibodies, respectively. (B) Anti-c-jun antibody shifts ILK-induced AP-1 complex. Nuclear extracts (2 μg) from HEK-293 cells transfected with ILK-V5 cDNA were incubated with 32P-AP-1 oligonucleotide in the absence or presence of anti-c-jun antibody or nonspecific IgG (10 μg). The latter did not induce a mobility shift of the complex (not shown).

Article Snippet: For the supershift assay, 10 μg of rabbit anti-c-jun antibody (Santa Cruz Biotechnology) or nonspecific IgG was added to the reaction mixture, subsequent to the addition of the 32 P-labeled oligonucleotide probe, and the mixture was incubated for 45 min at room temperature.

Techniques: Activity Assay, Electrophoretic Mobility Shift Assay, Transfection, Incubation, Labeling, Protein Binding, Western Blot, Expressing, Mobility Shift

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: Expression of p16INK4a (A) and pRB (B) in T-ALL cell lines. (A) Lysates from murine T-ALL cell lines were immunoprecipitated by using a monoclonal antibody that recognizes mouse p16INK4a or PAb 419 as a nonspecific control. Samples were resolved by SDS-PAGE and subjected to immunoblot analysis using the same p16INK4a antibody. (B) Lysates from murine T-ALL cells were subjected to immunoblot analysis with the anti-Rb antibody G3-245 (Pharmingen). Lysates from Balb/c 3T3 cells were used as a positive control. Both the hypo- and hyperphosphorylated forms of pRB can be seen in each sample. Size markers are indicated on the left.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Expressing, Immunoprecipitation, Control, SDS Page, Western Blot, Positive Control

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: Expression of hp16INK4a in MSCV-hp16INK4a-infected T-ALL cells (A) and binding of human p16INK4a to murine CDK4 (B). (A) Lysates from MSCV-hp16INK4a-infected and from uninfected T-ALL 4, T-ALL 5, and T-ALL 68 cells were immunoprecipitated with a polyclonal antibody that specifically recognizes human but not murine p16INK4a or with pAb 419 as a nonspecific control. Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblot analysis with the same p16INK4a antibody. Uninfected cells were used as negative controls and the human glioblastoma cell line D384 was used as a positive control for hp16INK4a. (B) Lysates from infected and uninfected T-ALL 68 cells were immunoprecipitated as in (A), and samples were subjected to immunoblot analysis with a monoclonal antibody that binds murine and human (weakly) CDK4. D384 was used as a control for binding of human CDK4 by human p16INK4a. Size markers are indicated on the left.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Expressing, Infection, Binding Assay, Immunoprecipitation, Control, SDS Page, Western Blot, Positive Control

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: MSCV-hp16INK4a suppresses the proliferation of infected murine T-ALL cell lines. T-ALL 4 (A), T-ALL 5 (B), and T-ALL 68 (C) were infected as described. The rate of proliferation was determined by labeling with [3H]-thymidine for 6 hours on the indicated days. The amount of [3H]-thymidine incorporated for each infected cell line is plotted versus the number of days after infection. The status of endogenous p16INK4a is indicated for each line.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Infection, Labeling

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: Expression of human p16INK4a in murine T-ALL cell lines induces G1-arrest. T-ALL 4 (A), T-ALL 5 (B), and T-ALL 68 (C) cells were infected as described. On days 1, 2, and 3 after infection, the fraction of cells in the G1- , S- , and G2-phases of the cell cycle was determined by BrdU incorporation and staining with PI. Those cells with a DNA content less than 2N as determined by PI staining were considered to be sub G1. The status of endogenous p16INK4a is indicated for each line.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Expressing, Infection, BrdU Incorporation Assay, Staining

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