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: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell Lines and Cell Culture Normal human fibroblast (GM00637), XPA deficient human fibroblast (GM04429), XPV deficient human fibroblast (GM03617) and mouse embryonic fibroblast cells were grown in DMEM medium (GenDEPOT) containing 10% fetal bovine serum (GenDEPOT) and 1% Antibiotic-Antimyocotic (GenDEPOT).

Techniques: Knock-Out, Staining, Transfection, Construct, Plasmid Preparation, Purification, DNA Ligation, DNA Purification, Proliferation Assay, Single Cell Gel Electrophoresis, Cell Culture, Software

Journal: Cell Death & Disease

Article Title: A role for TSPO in mitochondrial Ca 2+ homeostasis and redox stress signaling

doi: 10.1038/cddis.2017.186

Figure Lengend Snippet: TSPO re-routes cellular Ca 2+ signals in mammalian cells. ( a ) Representative Ca 2+ transients from MEFs transfected with mtAeq and stimulated with ATP (1 μ M). ( b ) Graph showing the mean max [Ca 2+ ] m n =5; ⩾50 cells per field of analysis; P <0.05. ( c ) Representative Ca 2+ transients from MEFs transfected with cytAeq and stimulated with ATP (1 mM). ( d ) Graph showing the mean max [Ca 2+ ] c n =3; ⩾50 cells per field of analysis; P <0.05. ( e ) Representative mitochondrial Ca 2+ traces obtained in MEFs loaded with Rhod-2 and stimulated with Thapsigargin (100 nM). ( f ) Graph showing mean maximum Rhod-2 fluorescence intensity in MEFs. n =4; ⩾40 cells per field of analysis; P <0.001. ( g ) Representative cytosolic Ca 2+ traces obtained in CF35 cells loaded with Fluo-4 and stimulated with Thapsigargin (100 nM). ( h ) Graph showing mean maximum Fluo-4 fluorescence intensity in MEFs ( n =3; ⩾40 cells per field of analysis; P <0.001). ( i ) Mean intensity of DHE fluorescence between control and TSPO-KD cells treated with Thapsigargin (* P <0.05, ** P <0.01, *** P <0.001)

Article Snippet: Mouse embryonic fibroblast cells (MEFs) , — a kind gift from Professor William Craigen (Bayer College of Medicine Medical Genetics Laboratories, Houston, TX, USA) and canine mammary gland epithelial cells (CF35), SH-SY5Y neuroblastoma (ATCC, Manassas, VA, USA) were maintained in a temperature-controlled, humidified incubator at 37 °C with 5% CO 2 (Hera Cell 240, Thermo Scientific, Essex, UK) in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, 11995065) supplemented with 10% fetal bovine serum (FBS; Life Technologies, 10082147), 100 U/ml penicillin and 100 mg/ml streptomycin (Life Technologies, 15140122).

Techniques: Transfection, Fluorescence, Control

Journal: Cell Death & Disease

Article Title: A role for TSPO in mitochondrial Ca 2+ homeostasis and redox stress signaling

doi: 10.1038/cddis.2017.186

Figure Lengend Snippet: TSPO-dependent Ca 2+ regulation occurs at the OMM and requires VDAC1 and PKA. ( a ) Real-time qRT-PCR studies show mRNA levels of MCU, normalized to levels of VDAC in MEFs; n =3; P >0.05. ( b ) Real-time qRT-PCR studies show mRNA levels of MCU, normalized to levels of VDAC in MEFs with modulated MCU expression; n =7; *** P <0.001. ( c ) Real-time qRT-PCR studies show mRNA levels of TSPO, normalized to levels of VDAC in MEFs with modulated MCU expression, n =5. ( d and e ) Immunoblot of MCU in MEFs WT and knocked out for the gene with quantification in e . ( f ) Representative traces showing ATP-induced mitochondrial Ca 2+ uptake in MEFs. ( g ) Graph showing the mean maximum [Ca 2+ ] m in response to ATP (1 mM) in MEFs ( n =3; *** P <0.001). ( h ) Representative traces showing ATP-induced Ca 2+ transients in TSPO-silenced VDAC1 −/− MEFs. ( i ) Bar chart showing the mean maximum [Ca 2+ ] m in VDAC1 −/− MEFs; n =4; ** P <0.05. ( j ) Representative traces showing ATP-induced Ca 2+ transients MEFs exposed to PKI

Article Snippet: Mouse embryonic fibroblast cells (MEFs) , — a kind gift from Professor William Craigen (Bayer College of Medicine Medical Genetics Laboratories, Houston, TX, USA) and canine mammary gland epithelial cells (CF35), SH-SY5Y neuroblastoma (ATCC, Manassas, VA, USA) were maintained in a temperature-controlled, humidified incubator at 37 °C with 5% CO 2 (Hera Cell 240, Thermo Scientific, Essex, UK) in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, 11995065) supplemented with 10% fetal bovine serum (FBS; Life Technologies, 10082147), 100 U/ml penicillin and 100 mg/ml streptomycin (Life Technologies, 15140122).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: A role for TSPO in mitochondrial Ca 2+ homeostasis and redox stress signaling

doi: 10.1038/cddis.2017.186

Figure Lengend Snippet: TSPO recruits ACBD3 and PKA to mitochondria and regulates VDAC1 phosphorylation. ( a ) Immunoblot of mitochondrial and ( b ) cytosolic extracts from control and +TSPO MEFs run in duplicate with antibodies indicated on the right. ( c ) Bar charts showing the mean band density of ACBD3, PKA, TSPO normalized to ATPB5 in mitochondrial fractions ( n =4) and ( d ) ACBD3, PKA normalized to ACTB in cytosolic fractions. ( e ) Immunoblot analysis of mitochondrial ACBD3 and PKA in SH-TSPOkd. ( f ) ACBD3, PKA, TSPO normalized to SDHA in mitochondrial fractions ( n =4). ( g ) Immunoprecipitation of VDAC from SH-SY5Y cells in TSPO-modulated conditions. The lower section (input 5%) western blotted with VDAC1 and TSPO antibodies. The upper section of the blot (IP) shows phosphorylated VDAC1. ( h ) Bar chart showing the mean band density of α -phosphoserine/threonine/tyrosine according to TSPO expression, normalized to input VDAC1 ( n =3; * P <0.05, ** P <0.01, *** P <0.001)

Article Snippet: Mouse embryonic fibroblast cells (MEFs) , — a kind gift from Professor William Craigen (Bayer College of Medicine Medical Genetics Laboratories, Houston, TX, USA) and canine mammary gland epithelial cells (CF35), SH-SY5Y neuroblastoma (ATCC, Manassas, VA, USA) were maintained in a temperature-controlled, humidified incubator at 37 °C with 5% CO 2 (Hera Cell 240, Thermo Scientific, Essex, UK) in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, 11995065) supplemented with 10% fetal bovine serum (FBS; Life Technologies, 10082147), 100 U/ml penicillin and 100 mg/ml streptomycin (Life Technologies, 15140122).

Techniques: Phospho-proteomics, Western Blot, Control, Immunoprecipitation, Expressing