Journal: Cancer research

Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells

doi: 10.1158/0008-5472.CAN-17-3953

Figure Lengend Snippet: Optimization of treatment schedule by combination of G9Ai and 5-aza-CdR in A2780 cells. A) Western blot analysis of H3K9me1 and H3K9me2 levels in response to increasing concentrations of UNC0638 in A2780 cells 48 h after treatment. Total histone H3 was used as a loading control. Representative blots from 3 independent experiments were shown. (B) Effects of dosing schedule by combination treatment with G9Ai and 5-aza-CdR on HERV-Fc1 expression. A2780 cells (2.5×105) were seeded in 100 mm dishes at d −1, treated with 400 nM G9Ai, 100 nM 5-aza-CdR or their combinations according to the schedule shown at the top panel. Cells were harvested at d 5 after 5-aza-CdR treatment. HERV-Fc1 expression levels were then assayed by quantitative RT-PCR using the expression levels of TATA-binding protein (TBP) as a loading control, and normalized to the level of HERV-Fc1 expression after 5-aza-CdR treatment alone. Values are presented as mean ± SEM of three independent experiments. A one-way repeated measures ANOVA was used for statistical analysis. *P <0.05.

Article Snippet: Antibodies against G9A (Perseus Proteomics Inc, PP-A8620A-00), Tubulin (Cell Signaling, 86298S), TBP (Santa Cruz Biotech sc-74596), H3K9me1 (Epicypher 13–0014), H3K9me2 (Abcam #1220), H3K9me3 (Active Motif #39161) and total H3 (Abcam #12079) were used.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Binding Assay

Journal: Cancer research

Article Title: Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian cancer cells

doi: 10.1158/0008-5472.CAN-17-3953

Figure Lengend Snippet: Changes in G9A protein and histone modification levels in A2780 cells at d 5 of the treatment schedule. A) Western blot analysis of G9A protein levels in whole cell, chromatin, and soluble fractions. TATA-binding protein (TBP) and beta tubulin proteins were used as controls. Representative blots were shown from three independent experiments. B) Bar graph shows the quantification of the chromatin-associated G9A protein levels relative to TBP protein levels. The values were normalized to the levels of G9A protein before treatment and are represented as mean ± SEM in three independent experiments. One-way repeated measures ANOVA with Geisser-Greenhouse’s epsilon correction was used for statistical analysis. *P <0.05; stars in black, blue and red indicate comparison to untreated, G9Ai treated, and 5-aza-CdR treated samples, respectively. C) Western blot analysis of total H3K9me1/2/3 modification levels in A2780 cells after treatments. Representative blots were shown from three independent experiments. D) Bar graphs show the quantification of H3K9me1/2/3 modification levels relative to total H3 levels and normalized to the levels in untreated samples in three independent experiments. Statistical analysis was performed as in B). E) Quantification of H3K9me2, H3K4me3 and H3K27ac levels by ChIP-qPCR at LTR regions of HERV-Fc1 (Fc1 LTRa and b) and MLT1N2. The intergenic spacer region of the 35S ribosomal DNA genes (IGS rDNA) and the promoter region of beta-actin (ACTBp) were used as controls. Values are mean ± SEM (n=3). One-way repeated measures ANOVA was used for statistical analysis. *P <0.05; stars in black, blue and red indicate comparison to untreated, G9Ai treated, and 5-aza-CdR treated samples, respectively.

Article Snippet: Antibodies against G9A (Perseus Proteomics Inc, PP-A8620A-00), Tubulin (Cell Signaling, 86298S), TBP (Santa Cruz Biotech sc-74596), H3K9me1 (Epicypher 13–0014), H3K9me2 (Abcam #1220), H3K9me3 (Active Motif #39161) and total H3 (Abcam #12079) were used.

Techniques: Modification, Western Blot, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: PERK Utilizes Intrinsic Lipid Kinase Activity To Generate Phosphatidic Acid, Mediate Akt Activation, and Promote Adipocyte Differentiation

doi: 10.1128/MCB.00063-12

Figure Lengend Snippet: The ER stress sensor PERK demonstrates lipid kinase activity in vitro and in vivo. (a) PERK was immunopurified from p110+/+ or p110α−/− MEFs, and lipid kinase activity was determined in an in vitro assay with phosphatidylinositol as a substrate. NRS, nonspecific rabbit serum. Thaps, thapsigargin. (b) In vitro lipid kinase assay with phosphatidylinositol as a substrate and recombinant catalytic domain of PERK (GST-ΔNPERK) or kinase-dead PERK (K618A). (c) In vitro lipid kinase assay with phosphatidylinositol as a substrate and recombinant catalytic domain of PERK (GST-ΔNPERK), kinase-dead PERK (K618A), p38, GSK3β, PKA C (500 ng of each kinase), and PI3K p85/p110 complex. (d) In vitro lipid kinase assay with phosphatidylinositol as a substrate and recombinant catalytic domain of PERK (GST-ΔNPERK) or recombinant PI3K and various phosphoinositide substrates. CBE, crude brain extract. Products were resolved by chromatography with propanol and acetic acid as the mobile phase. (e) The radioactive spot from the TLC plate in panel d (with CBE as a substrate) was excised, and PERK product was deacylated and then resolved by HPLC with 3H-labeled PI, PI4P, and PI4,5P2 as standards. (f) In vitro lipid kinase assay with diacylglycerol (DAG) as a substrate and recombinant catalytic domain of PERK (GST-ΔNPERK). DAGC12, 1,2-dilauroyl-sn-glycerol. (g) In vitro lipid kinase assay with phosphatidylinositol (PI) or diacylglycerol (DAG) as a substrate and the recombinant catalytic domain of PERK in the presence or absence of recombinant PI3K p85α or with recombinant diacylglycerol kinase (DGK). DAGC14, 1,2-dimyristoyl-sn-glycerol; DAGC16, 1,2-dipalmitoyl-sn-glycerol; DAGC18, 1,2-distearoyl-sn-glycerol.

Article Snippet: 1,2-Dilauroyl-sn-glycerol (DAGC12), 1,2-dimyristoyl-sn-glycerol (DAGC14), 1,2-dipalmitoyl-sn-glycerol (DAG16), and 1,2-distearoyl-sn-glycerol (DAG18) were from Echelon Biosciences.

Techniques: Activity Assay, In Vitro, In Vivo, Kinase Assay, Recombinant, Chromatography, Labeling