Journal: Cancer research

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity

doi: 10.1158/0008-5472.CAN-16-0298

Figure Lengend Snippet: (A) Non-synonymous somatic IDH2, TET2, PIK3CA, and PIK3R1 mutations identified in the 13 SPCRPs studied here by massively parallel sequencing (WES or MSK-IMPACT), SNaPshot or Sanger sequencing. (B) Mutation plot shows the domain structure of IDH2 and the non-synonymous IDH2 mutations identified in SPCRP and in common forms of breast cancer published by TCGA available from cBioPortal (28). (C) 2HG analysis in IDH2-mutant SPCRP (case 12) and IDH wild-type invasive ductal carcinoma of no special type (IDC). (D) IDH2/TET2-mutant SPCRPs display global DNA hypermethylation assessed by Infinium MethylationEPIC BeadChip as compared to IDH2/TET2 wild-type IDCs (left). Unsupervised hierarchical clustering using all methylation values/ genes of six IDH2/TET2-mutant SPCRPs and two IDH2/TET2 wild-type IDCs using complete linkage and Euclidean distance (right). The 1000 genes with the highest variance across samples are displayed. Methylation status is color coded according to the legend. (E) H3K27me3 immunohistochemical analysis of four IDH2-mutant SPCRPs (top row) and four IDH2 wild-type IDCs (bottom row). Nuclear immunoreactivity for H3K27me3 was quantified using the H-score. *, p<0.05; Mann-Whitney U test.

Article Snippet: DNA transfections and analysis of transgene expression The human IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"588282795","term_text":"NM_002168"}} NM_002168 ) cDNA ORF clone pCMV6-IDH2::Myc-DDK was purchased from Origene (RC201152), and the R172S mutation introduced using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s recommendations.

Techniques: Sequencing, Mutagenesis, Methylation, Immunohistochemical staining, MANN-WHITNEY

Journal: Cancer research

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity

doi: 10.1158/0008-5472.CAN-16-0298

Figure Lengend Snippet: (A) Detection of 2HG as a readout of IDH2WT and IDH2R172S enzymatic activity in total protein lysates (left) and in conditioned media (right) of MCF10AP and MCF10AH1047R cells expressing empty vector control, IDH2WT or IDH2R172S. The same cell lysates from MCF10AP and MCF10AH1047R cells were analyzed for IDH2 protein expression by western blotting. Total tubulin was used as loading control; ns: not significant; ***P<0.001; ****P<0.0001; error bars represent standard deviation of mean. (B) Whole cell lysates of MCF10AP and MCF10AH1047R cells were analyzed for IDH2, total and phosphorylated RB, E-cadherin, and tubulin protein expression by western blotting (left), and quantified using near-infrared detection (LI-COR; Odyssey) (right); error bars represent standard deviation of mean.

Article Snippet: DNA transfections and analysis of transgene expression The human IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"588282795","term_text":"NM_002168"}} NM_002168 ) cDNA ORF clone pCMV6-IDH2::Myc-DDK was purchased from Origene (RC201152), and the R172S mutation introduced using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s recommendations.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Western Blot, Standard Deviation