paraffin embedded tissue microarray sections  (Proteintech)

Journal: International Journal of Biological Sciences

Article Title: DNMT3a promotes LUAD cell proliferation and metastasis by activating the HDAC7 signalling pathway

doi: 10.7150/ijbs.96509

Figure Lengend Snippet: High expression of HDAC7 predicts poor prognosis in LUAD. a Kaplan-Meier survival analysis of patients represented in the Kaplan-Meier Plotter database stratified by HDAC7 expression. b Representative images of HDAC7 IHC staining in LUAD and adjacent nontumour tissues. Scale bars, 200 μm and 20 μm (inset). c Statistical analysis of HDAC7 expression based on IHC staining in tumour tissue and adjacent nontumour tissue of 119 LUAD patients. Statistical analysis of HDAC7 expression based on IHC staining of samples from 119 LUAD patients stratified into subgroups of T classification, N classification and clinical stage. Kaplan-Meier survival analysis of 119 LUAD patients based on HDAC7 expression. d Comprehensive Kaplan-Meier survival analysis of 119 LUAD patients stratified by DNMT3a and HDAC7 expression based on tissue microarray IHC results. e Correlation analysis of DNMT3a and HDAC7 expression in LUAD tissues. f Representative images and statistical analysis of the colony formation assay. Colonies were visualized by crystal violet staining. Representative images and statistical analysis of the EdU incorporation assay. The results were calculated as the ratio of the number of EdU-positive cells (red fluorescence) to the total number of Hoechst 33342-positive cells (blue fluorescence). Scale bar, 100 μm (inset). g Representative wound healing assay images and results. The migration ability was quantified as the mean scratch area at each time point. The initial scratch area (0 h) was set as 100%. Scale bars, 100 μm (inset). h Representative transwell migration assay images and results. Scale bars, 200 μm (inset). * p < 0.05. ** p < 0.01.

Article Snippet: According to standard practice, immunohistochemical (IHC) staining of paraffin-embedded tissue microarray sections was performed using primary antibodies of anti-DNMT3a (1:400, 20954-1-AP, Proteintech) and anti-HDAC7 (1:100, #33418, Cell Signaling Technology (CST)).

Techniques: Expressing, Immunohistochemistry, Microarray, Colony Assay, Staining, Fluorescence, Wound Healing Assay, Migration, Transwell Migration Assay

Journal: EMBO Molecular Medicine

Article Title: Inhibition of DNA methylation promotes breast tumor sensitivity to netrin‐1 interference

doi: 10.15252/emmm.201505945

Figure Lengend Snippet: Bisulfite PCR sequencing (4 CpGs analyzed, region: light gray boxes in Fig A) indicated that DNA methylation of the NTN1 CpG island (CGi) was inversely correlated with its expression. Pearson correlation, P = 0.008, r = −0.55, n = 18. Bisulfite PCR sequencing (3 CpGs analyzed, region: light gray boxes in Fig A) indicated that DNA methylation of the DAPK1 CGi was inversely correlated with its expression. Pearson correlation, P = 0.003, r = −0.66, n = 22. Tissue microarrays (70 paraffin embedded sections) from human breast carcinomas were immuno‐stained with antibodies against DAPK1, UNC5B, and netrin‐1. Samples were classified in quartiles according to the level of netrin‐1 expression. The levels of DAPK1 and UNC5B expression (index constructed from the percentage of sections exhibiting a positive staining) in the first and fourth quartiles of netrin‐1 expressing groups were compared using a chi‐squared test. Representative staining corresponding to low and high levels of expression is shown for each antibody, and expression levels were determined from the percentage of sections exhibiting a positive staining. As a control, staining of the samples using a non‐related isotype antibody was performed. Quantification of the presence or absence of alterations in gene A was associated with the presence or absence of alterations in gene B in human breast tumors which was determined from cBioPortal web site, z ‐score threshold ± 2.2 Individual methylation plots. Red rectangles represent methylated CpG and blue rectangles unmethylated CpG. Mean DNA methylation inhibition of DAPK1 and NTN1 in MDA‐MB‐231 and HMLER cells upon DAC treatment.

Article Snippet: Tissue microarrays of paraffin embedded breast tumor sections were obtained from Super Bio Chips (Cliniscience, Nanterre, France).

Techniques: Sequencing, DNA Methylation Assay, Expressing, Staining, Construct, Methylation, Inhibition