Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes a transcriptional downregulation of RAD51 and other proteins known to control DSB repair by HR. RNA-seq was performed on U2OS cells transfected with non-silencing (NS), NFKB2 or RELB siRNA. ( A ) Knockdown is efficient for both p100/52 and RelB, based on mRNA measurements by RNA-seq (left) and protein level by western blot (right). ( B ) KEGG pathway analysis of was performed to predict the 10 most upregulated and 10 most downregulated pathways in response to siNFKB2. Corresponding effects in response to siRELB are also displayed. ( C ) Expression levels are displayed for 39 genes with known relevance to HR, as defined by the KEGG database. Genes are arrayed from left to right based on degree of expression change after siNFKB2 (* denotes P < 0.01).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Control, RNA Sequencing, Transfection, Knockdown, Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes downregulation of RAD51 protein. ( A ) Representative western blots demonstrate that transcriptional silencing of key NF-κB proteins leads to reduced levels of RAD51 protein. ( B ) Quantitation of western blots from three independent experiments demonstrate that RAD51 reductions after siNFKB2 are reproducible (error bars denote the SEM). ( C ) Comparable levels of RAD51 knockdown are observed using three different siRNAs (each at 25 nM) targeting NFKB2 in U2OS cells. ( D ) Forced overexpression of the p52 fragment of Nfkb2 in mouse lung tissue is associated with increased Rad51 expression (error bars denote standard error, * denotes P < 0.02, NS denotes non-silencing control).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Western Blot, Quantitation Assay, Knockdown, Over Expression, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 inhibits DNA damage-induced RAD51 focus formation in human cancer cells. Cells previously transfected with siRNA or non-silencing (NS) controls were pulse-labeled with EdU to mark S-phase cells and concomitantly treated with 30 nM CPT for 1 hour. Cells were harvested and immune-stained as indicated. Representative microscopic images of unselected nuclei ( A ) are displayed for cells collected 3-hours after CPT treatment, and ( B ) quantitation of RAD51 foci per nucleus is plotted for each of the indicated conditions. ( C ) Quantitation of EdU intensity in CPT-untreated cells is used to estimate cell cycle at the time of the EdU pulse. ( D ) A schematic representation of the treatment timeline is displayed. ( E ) The mean number of RAD51 foci per EdU-positive nucleus is plotted as a function of time following CPT treatment (error bars denote SEM for at least 100 nuclei within a single experimental replicate, * denotes P < 0.05, *** denotes P < 1e−13, and ns denotes P > 0.05).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Transfection, Labeling, Staining, Quantitation Assay

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: ( a ): Baseline clinical and pathological characteristics of bladder cancer patients. ( b ): Median and IQR of patients’ age and follow-up duration. ( c ): Expression intensity of SPP1.

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Expression pattern of SPP1 in pan-cancer. ( A ) The expression of the SPP1 mRNA level in multiple TCGA cancers and matching normal tissues. p < 0.001, except Thym cancer ( p = 0.7) and KICH cancer ( p = 0.53). ( B ) Increased mRNA expression level of SPP1 in bladder cancer. ( C ) Promoter methylation status of SPP1 in bladder cancer and matching normal tissues. All data were analyzed using the UALCAN web tool.

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing, Methylation

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Molecular alterations of SPP1 in cancers. ( A ) High amplifications/mutations of the SPP1 gene in bladder cancer compared other cancer types. ( B ) Positions and mutation frequency in SPP1 in bladder cancer.

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Mutagenesis

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Cytoplasmic expression of SPP1 in bladder carcinoma. Immunohistochemical staining of the bladder cancer tissue microarray using an SPP1 antibody. Figures showing: no expression ( A , B ), weak ( C , D ), moderate ( E , F ) and strong expression ( G , H ) of SPP1. Images were taken using 10× and 40× magnification objectives (scale bar equals 1 mm).

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Nuclear SPP1 expression in bladder carcinoma. Immunohistochemical staining of the bladder cancer tissue microarray using an SPP1 antibody. Figures showing: no expression ( C , D ), and strong expression of SPP1 ( A , B ). Images were taken with 10× and 40× magnification objectives (scale bar equals 1 mm).

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Correlation between cytoplasmic SPP1 expression and patients’ clinicopathological characteristics.

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: SPP1 expression and patients’ survival. Kaplan–Meier survival curve for bladder cancer patients expressing cytoplasmic ( A ) and nuclear ( B ) patterns of SPP1 (low expression vs. high expression). Low SPP1 immunostaining is associated with poor overall survival (log-rank p = 0.022).

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing, Immunostaining

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Enrichment analysis of SPP1 in bladder cancer. ( A ) Identification of SPP1-interacting genes. Protein–protein interaction map and hub genes of SPP1. The size of the hub is proportional to the expression level. ( B ) Identification of the SPP1 co-expression network. The figure was generated using the online cBioPortal database. ( C ) KEGG functional enrichment analysis of SPP1. The figure was generated using the online cBioPortal database.

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing, Generated, Functional Assay

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Correlation between immune cells and SPP1 expression. TIMER analysis of the correlation between SPP1 expression and immune cells’ infiltration. Purity-adjusted Spearman’s rho across various cell types by different algorithms.

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing

Journal: Cancers

Article Title: Identification of SPP1 as a Prognostic Biomarker and Immune Cells Modulator in Urothelial Bladder Cancer: A Bioinformatics Analysis

doi: 10.3390/cancers15235704

Figure Lengend Snippet: Relationship between SPP1 expression and immune checkpoint genes in bladder cancer. ( A ) Correlation analysis between SPP1 expression and immune checkpoint genes. ( B ) The expression of immune checkpoint genes in relation to SPP1 expression. Data were analyzed using the cBioPortal cancer genomics website on TCGA data. The p -value significance codes: *** ≤0.001, ** ≤0.01, * ≤0.05.

Article Snippet: The slides were then incubated with the primary polyclonal human anti-SPP1 antibody (Spring Bioscience, Pleasanton, CA, USA. cat#E3284) at a dilution of 1:100 for 32 min.

Techniques: Expressing