Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) The protein levels of CPF components CSTF2, CPSF3, SYMPK, Xrn2, PPP1CB and PPP1CC in AFF4 depletion and AFF4 mutation HCT116 cells. α-Tubulin was used as a loading control. (B) Schematic diagram shows the primers sites around FOS gene. ChIP-qPCR analysis showing the occupancy of CSTF2 at FOS gene in AFF4 depletion and AFF4 mutation serum induced HCT116 cells. The HEMO gene acts as a negative control for ChIP-qPCR. (C) RT-qPCR analysis of CSTF2 gene expression after CSTF2 knockdown in wildtype and AFF4 mutation HCT116 cell lines. (D) RT-qPCR analysis of FOS gene expression after CSTF2 knockdown in wildtype and AFF4 mutation HCT116 cell lines. (E) ChIP-qPCR showing the occupancy of Pol II after CSTF2 knockdown in AFF4 mut. The HEMO gene acts as a negative control for ChIP-qPCR.

Article Snippet: Ser2P Pol II (ab5095), Ser5P Pol II (ab5131), PAF1 (ab20662) rabbit polyclonal antibody were purchased from Abcam; SPT5 (A9193), SPT6 (A16434), CSTF2 (A8116), CPSF3 (A12368), SYMPK (A8722), Xrn2 (A18350), PPP1CB (A13528), PPP1CC (A4025) rabbit polyclonal antibody were purchased from ABclonal.

Techniques: Mutagenesis, Negative Control, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) The protein levels of CPF components CSTF2, CPSF3, SYMPK, Xrn2, PPP1CB and PPP1CC in AFF4 depletion and AFF4 mutation serum induced HCT116 cells. α-Tubulin was used as a loading control. (B) RT-qPCR showing the RNA expression level of CPSF3 after CPSF3 knockdown in wildtype and AFF4 MT cells. (C) RT-qPCR showing the RNA expression level of FOS after CPSF3 knockdown in wildtype and AFF4 MT cells. (D) The change of Pol II enrichment around the FOS gene after knockdown CPSF3 in WT and AFF4 MT cells analyzed by ChIP-qPCR. The HEMO gene acts as a negative control for ChIP-qPCR.

Article Snippet: Ser2P Pol II (ab5095), Ser5P Pol II (ab5131), PAF1 (ab20662) rabbit polyclonal antibody were purchased from Abcam; SPT5 (A9193), SPT6 (A16434), CSTF2 (A8116), CPSF3 (A12368), SYMPK (A8722), Xrn2 (A18350), PPP1CB (A13528), PPP1CC (A4025) rabbit polyclonal antibody were purchased from ABclonal.

Techniques: Mutagenesis, Quantitative RT-PCR, RNA Expression, Negative Control

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: DLGAP5 expression levels in breast tissues of patients with BC. (A) mRNA expression levels of DLGAP5 in BC tissues compared with normal breast samples. (B) mRNA expression levels of DLGAP5 in an expanded sample size following analysis of the GSE29431 and GSE61304 datasets. (C and D) ROC curve based on DLGAP5 expression levels in (C) GSE29431 and (D) GSE61304 datasets for predicting breast cancer tissue classification. (E) PFS and (F) OS analysis of patients with BC with low and high DLGAP5 expression levels. ***P<0.001. DLGAP5, discs large-associated protein 5; PFS, progression-free survival; OS, overall survival; BC, breast cancer; ROC, receiver operating characteristic; HR, hazard ratio.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Parameters associated with DLGAP5 expression in the GOBO database in patients with breast cancer. (A) Boxplot of DLGAP5 mRNA expression levels stratified based on the molecular subtypes. Boxplot of DLGAP5 mRNA expression levels stratified by (B) PAM50 classification, (C) estrogen receptor status and (D) tumor grade. The expression levels of DLGAP5 were compared among subtypes of breast cancer. P<0.00001 vs. basal, ER-neg and grade 1. DLGAP5, discs large-associated protein 5. HER2, human epidermal growth factor receptor-2; LumA, luminal A subtype; LumB, luminal B subtype; ER, estrogen receptor; -neg, negative; -pos, positive.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Representative immunohistochemistry images of DLGAP5 expression levels in breast cancer tissue samples. (A and B) Strong, weak and null expression levels of DLGAP5 in (A) breast tumor tissue (n=160) and (B) normal breast tissue (n=32). Magnification, ×400. DLGAP5, discs large-associated protein 5.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Immunohistochemistry, Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Association of DLGAP5 expression levels with clinical characteristics of patients with breast cancer.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Underlying molecular mechanisms of DLGAP5 activity in breast cancer. (A) Different GO enriched terms associated with genes co-expressed with DLGAP5 in breast cancer. (B) KEGG pathway enrichment of the co-expressed genes. (C) Subcellular localization of DLGAP5. The green intensity represents the localization probability. (D) Domain demonstration of DLGAP5 and its LIR motif (in red) exhibiting conservation across species. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DLGAP5, discs large-associated protein 5.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Activity Assay

Journal: Oncology Letters

Article Title: Elevated mRNA expression levels of DLGAP5 are associated with poor prognosis in breast cancer

doi: 10.3892/ol.2020.11533

Figure Lengend Snippet: Effects of DLGAP5 on MDA-MB-231 cell proliferation. (A) Expression levels of DLGAP5 in MDA-MB-231 cells transfected with si-NC, si-DLGAP5-1 and si-DLGAP5-2 was determined by reverse transcription-quantitative PCR after transfection for 48 h. (B) Cell Counting Kit-8 assay was used to detect the effects of DLGAP5 on MDA-MB-231 cell proliferation. (C and D) Flow cytometry was used to detect the effects of DLGAP5 on cell cycle regulation in MDA-MB-231 cells. *P<0.05 and ***P<0.001 vs. si-NC; **P<0.01 si-DLGAP5-1 vs. si-NC; ### P<0.001 si-DLGAP5-2 vs. si-NC. si, small interfering; NC, negative control; DLGAP5, discs large-associated protein 5.

Article Snippet: Antigens were retrieved in citrate buffer at 95°C (pH 6) for 15 min, and 3% hydrogen peroxide was used for endogenous peroxidase blocking at 37°C for 30 min, followed by incubation with 10% goat serum (Abcam) at room temperature for 1 h. IHC was performed using a rabbit polyclonal anti-human DLGAP5 primary antibody (1:200; cat. no. A13575; Abclonal), and the slides were incubated overnight at 4°C.

Techniques: Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Flow Cytometry, Negative Control