Journal: eLife

Article Title: Unleashing a novel function of Endonuclease G in mitochondrial genome instability

doi: 10.7554/eLife.69916

Figure Lengend Snippet: ( A ) Localization of Endonuclease G in mitochondria in different cell lines. Representative images of localization of Endonuclease G to mitochondria in HeLa, MEF and HEK293T cells. FITC-conjugated secondary antibodies were used for detecting Endonuclease G proteins. MtDR is Mitotracker Red, a mitochondrial marker. DAPI is used as nuclear stain. ( B ) Colocalization analyses of Endonuclease G and Mitotracker signals using JaCoP in ImageJ software based on immunofluorescence studies performed in multiple cell lines (see ). Minimum of 50 cells were used for analysis of colocalization of red and green signals and plotted as the colocalization value of green overlapping red. Y- axis depicts the Mander’s colocalization coefficient value calculated for green over red and plotted in the form of dot plot. The significance was calculated using GraphPad Prism 5.0 with respect to secondary control and shown as mean ± SEM ( ns not significant, *p<0.05, **p<0.005, ***p<0.0001). ( C ) Representative images of colocalization of Endonuclease G to mitochondrial matrix (TFAM) in HeLa cells. Conjugated secondary antibodies were used for detecting Endonuclease G (Alexa Fluor 488) and mitochondrial matrix protein, TFAM (Alexa Fluor 568). DAPI is used as nuclear stain. ( D ) Colocalization analyses of Endonuclease G and TFAM signals using JaCoP in ImageJ software. Minimum of 50 cells were used for analysis of colocalization of red and green signals and plotted. Y-axis depicts the Mander’s colocalization coefficient value calculated for green over red and plotted in the form of dot plot. Control represents the panel where only one of the primary antibodies was used. ( E ) Knockdown of Endonuclease G from HeLa cell using PEI mediated transfection. shRNA against Endonuclease G cloned plasmid was used for transfection. Cells were harvested after 48 h and mitochondrial extracts were prepared. Western blotting was performed to confirm the knockdown of Endonuclease G from the HeLa cells. Actin served as a loading control. ( F ) The knockdown extract was incubated with the plasmid and used for the primer extension using VKK11 primer (‘I’ and ‘II’ are two biological repeats). Extract prepared from the sample transfected with scrambled plasmid served as a control (SCR control). Lanes 3, 4, 7, and 8 serve as scrambled controls while lanes 5, 6, 9, and 10 are for knockdown extracts. I and II represent two independent batches of experiments. ‘M’ is a 50 nt ladder. ( G ) Bar diagram representing the cleavage intensity of the extracts prepared after transfection with scrambled plasmid and shEndo G plasmid. In panels F and G, a minimum of three biological repeats were performed and the data is shown with the error bar calculated as SEM (ns: not significant, *p<0.05, **p<0.005, ***p<0.0001). Refer also . Figure 8—source data 1. Localization of Endonuclease G to mitochondria. Figure 8—source data 2. Immunofluorescence showing the Localization of Endonuclease G to mitochondria.

Article Snippet: HeLa (human cervical cancer), HCT116 (human colon cancer), MEF (mouse embryonic fibroblast) and HEK 293T (human embryonic kidney epithelial cell line) were purchased from National Centre for Cell Science, Pune, India.

Techniques: Marker, Staining, Software, Immunofluorescence, Control, Knockdown, Transfection, shRNA, Clone Assay, Plasmid Preparation, Western Blot, Incubation

Journal: Theranostics

Article Title: High OGT activity is essential for MYC-driven proliferation of prostate cancer cells

doi: 10.7150/thno.30834

Figure Lengend Snippet: Identification of transcription factors that bind to O-GlcNAc marked chromatin. A) Distribution of differently modified histone 3 reads within ±2kb of O-GlcNAc peak. B) . The top 3 motifs enriched for the O-GlcNAc ChIP-seq data showing similarity with known previously identified transcription factor motifs from published datasets. C) O-GlcNAc ChIP-seq consensus overlap with MYC ChIP-seq data reported in this study. D) O-GlcNAc ChIP-seq consensus overlap with MYC ChIP-exo dataset reported previously (GSE73994). E) OSMI-2 disrupts interaction between MYC and HCF-1. Immunoprecipitation (IP) of MYC. IgG denotes negative control. MYC was overexpressed by addition of doxycycline using the LNCaP-MYC cell line for 4 hours either in the presence or absence of 40µM OSMI-2. Experiment was repeated four times. F) Knockout of OGT disrupts the interaction between MYC and HCF-1. Experiment was performed in a mouse embryonic fibroblast cell line that has been genetically engineered to enable removal of OGT gene by addition of 0.5µM Tamoxifen (Tam). After two days of DMSO- or Tam-treatments, cell lysates were prepared and used for immunoprecipitation. Data shown is representative of two biological replicates. G) Overlap of O-GlcNAc (this study), MYC (this study) and HCF-1 (ENCSR000ECH) ChIP-seq data.

Article Snippet: Tamoxifen-inducible OGT knockout mouse embryonic fibroblast (MEF) cell line was obtained from Dr. Natasha Zachara at the CardioPEG CoreC4 (NHLBI P01 HL107153) at Johns Hopkins University School of Medicine .

Techniques: Modification, ChIP-sequencing, Immunoprecipitation, Negative Control, Knock-Out

Journal: bioRxiv

Article Title: Cross-species blastocyst chimerism between nonhuman primates using iPSCs

doi: 10.1101/635250

Figure Lengend Snippet: A) Colony morphology of pig-tailed macaque iPSC in NHP-E8X media on MEF at 4X and 10X resolution (left) and the colony morphology of pig-tailed macaque iPSCs in E8 media on MEF at 4X and 10X resolution (right). B) Co-culture of human-human iPSC (top-left) and human-chimp iPSC (top-right). Human and chimpanzee iPSCs mix and integrate homogenously without forming segregated colonies. Co-culture of mouse-mouse iPSCs form mixed and homogenously integrated colonies (bottom-left). Human and mouse iPSCs form separate and segregated colonies (bottom-right). C) Cardiomyocyte layers differentiated from the co-culture mixture of chimpanzee and human iPSCs. The tdTomato-fluorescent labeled beating chimpanzee iPSC (red, left) and the human GFP-fluorescent labeled beating cardiomyocyte (green, middle) and the overlay image of the mixed cardiomyocytes from the mixture of chimpanzee and human iPSC (Supplementary video 1). Electrocardiogram of cardiomyocyte derived from the mixture of human and chimpanzee iPSCs indicating contraction and relaxation start, peak, and end (right). D) Hematoxylin and eosin (H&E)-stained sections of chimpanzee teratoma showing all three embryonic germ layers, derived from chimpanzee iPSCs with and without Bcl2 transduction. Ectoderm (a’, b’) is characterized by the presence of neural ectoderm arranged in rosette-like patterns (black arrows, a’ and b’), that resemble embryonic neural tubes (white arrows, a’). Endoderm (a’’, b’’) is characterized by the presence of glandular and/or secretory epithelium (black asterisks). Mesoderm (a’’’, b’’’) is characterized by the presence of elongate, mesenchymal cells (black arrows, a’’’) and cells embedded in a pale blue gray hyaline matrix resembling articular cartilage (black asterisk, b’’’). Magnification: 40x. Scale bars: 20μm.

Article Snippet: On the day after transfection (day 1), all the cells transfected with Sendai virus were transferred into a 6-well plate (30,000 cells per well) each well containing ~150,000 mouse embryonic fibroblast (MEF) feeder cells (Stemcell Technologies Inc. Vancouver, Canada).

Techniques: Co-Culture Assay, Labeling, Derivative Assay, Staining, Transduction