Journal: The Journal of Biological Chemistry

Article Title: Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors *

doi: 10.1074/jbc.M113.507152

Figure Lengend Snippet: Expression of CYP2C is inducible by light and targeted knockdown of CYP2C55 rescues light-induced cell death. Deduced amino acids of encoded human CYP2C9 and mouse CYP2C55 and CYP2C29 enzymes were aligned using Clustal Omega. Rectangular boxes represent key conserved residues for catalytic activity (gray), ligand binding (black), and heme stabilization (dashed) (see Ref. 90) (A). 661W cells on 6-well plates with/without sulfaphenazole pretreatment for 2 h at 37 °C were exposed to light (L) or remained in the dark (D). Expression of CYP2C proteins was detected from prepared cell lysates by Western blotting using anti-human CYP2C9 polyclonal antibody that cross-reacts with mouse CYP2C isozymes. The housekeeping gene β-actin was used as a loading control. Relative expression is presented by normalization with the densitometric ratio of CYP2C/β-actin in dark control cells (B). 661W cells at 50% confluence were transfected with Silencer Select predesigned siRNAs targeted to mouse CYP2C55 or CYP2C29 or mock-transfected with the scrambled control (Con.) oligos (n = 4/sample group). Cell lysates were prepared at 48 h after transfection to assess silencing efficiency by Western blotting. The densitometric ratio of CYP2C55 or CYP2C29/β-actin is presented (C). Cellular ATP indicative of cell viability was measured after light exposure (n = 4/sample group) (D). **, p < 0.01; *, p < 0.05; n.s., not significant. RLU, relative light units. In B, bars represent the relative ratio of densitometry (CYP2C/β-actin) from one representative experiment. In C, bars represent the absolute ratio of densitometry (siCYP2C55 or siCYP2C29/β-actin).

Article Snippet: Generation of Stably Transfected CYP2C9-GFP Cell Lines in 661W Cells 661W cells at 50% confluence on 96-well plates were transfected with pCMV6-CYP2C9-GFP PrecisionShuttle mammalian expression vector (0.2 μg/well) using Turbofectin 8.0 (OriGene).

Techniques: Expressing, Activity Assay, Ligand Binding Assay, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors *

doi: 10.1074/jbc.M113.507152

Figure Lengend Snippet: Stable expression of functional human CYP2C9-GFP fusion protein enhances cellular sensitivity to light. pCMV6-CYP2C9-GFP stably transfected 661W cells were examined by confocal microscopy showing the bright field and its switch to FITC mode to assess homogeneity of GFP-positive cells. The arrow indicates expression of CYP2C9-GFP on the surface of the cell body (A). The enzymatic activity of CYP2C9-GFP fusion protein with/without sulfaphenazole (10 μm) pretreatment was examined by a cell-based luminogenic assay that generates luminogenic signal from CYP2C9-catalyzed oxidation of the selective substrate luciferin-H. Wild-type (WT) 661W cells were controls. n = 3/luciferin-H concentration. ***, p < 0.001; *, p < 0.05 versus CYP2C9-GFP + SFZ (B). pCMV6-CYP2C9-GFP stably transfected 661W cells with/without pretreatment with sulfaphenazole (10 μm) (n = 12/treatment or non-treatment group) and wild-type cells (n = 24) were exposed to light for 4 h. Cellular ATP indicative of cell viability was measured after light (L) exposure (C). n.s., not significant. Error bars represent means ± S.D. RLU, relative light units; D, dark.

Article Snippet: Generation of Stably Transfected CYP2C9-GFP Cell Lines in 661W Cells 661W cells at 50% confluence on 96-well plates were transfected with pCMV6-CYP2C9-GFP PrecisionShuttle mammalian expression vector (0.2 μg/well) using Turbofectin 8.0 (OriGene).

Techniques: Expressing, Functional Assay, Stable Transfection, Transfection, Confocal Microscopy, Activity Assay, Concentration Assay

Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) Screnshoots of genome windows showing co-occurrence between TDP43 (red), DRIPc-seq (green), untreated (dark blue) and RNH-treated (light blue) DRIP-seq and RNA-seq (yellow) data. Genome location is indicated in the top left corner and scale in the top right. Coverage scale indicated in the top left of each track. Biological replicates are also indicated (R1, R2). B) Venn diagram showing correlation between genes bind by TDP-43 (TDP43; red circle), genes forming R loops (DRIPc-seq; green) and expressed genes (RNA-seq; yellow). Numbers refer to genes co-occurring between conditions. C) TDP-43 ChIP-seq average coverage (log10) across silent (-) and expressed (+) genes. ****, P < 0,0001 (Mann-Whitney U test, two-tailed). D) Metagene analysis showing TDP-43 ChIP-seq coverage (red line) along gene body (+/- 2kb) of silent (-) and expressed (+) genes. Mean coverage is plotted in the upper panel and heatmap intensities for the entire gene population is shown below. Scale is also indicated.

Article Snippet: SH-SY5Y cells were stably transfected using Fugene HD Transfection Reagent (Promega) with C-terminally GFP-tagged TDP-43 WT vector (PS100010 pCMV6-AC-GFP), C-terminally GFP-tagged TDP-43 A382T mutation vector (CW303334 mutated ORF of RC210639 at nt position 1144, changed from G to A, to obtain A382T, inserted in PS100001) and a C-terminally GFP-tagged TDP-43 G294V mutation vector (CW303287 mutated ORF of RG210639 at nt position 881, changed from G to T, to obtain G294V, inserted in PS100010), purchased from OriGene.

Techniques: RNA Sequencing Assay, ChIP-sequencing, MANN-WHITNEY, Two Tailed Test

Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) IF of LCL-CTL, LCL-TDP382, LCL-SALS using an anti-TDP-43 antibody and an anti-S9.6 antibody after methanol fixation. The scatter plots show the increase of S9.6 signal intensity and the decrease in TDP-43 nuclear content in SH-TDP382. Median values are indicated. Scale bar: 25μm. *, P< 0,05 (Mann-Whitney U test, two-tailed). When no asterisk is shown indicates that is not significant. B) Flow cytometry plot reports the amount of R-loops in LCLs (blue) in comparison to LCL-TDP382 (orange) and LCL-SALS (blue). The RNaseH action on LCL-TDP382 decreases the presence of R-loops signal (orange peak vs green peak). Histogram shows S9.6 mean fluorescence of LCL-CTL, LCL-TDP382, LCL-SALS in presence (+) and in absence (-) of RNaseH. The value represented is the mean ± SEM of three biological experiments. ANOVA, Newman-Keuls Multiple Comparison Test, *P <0,05. When no asterisk is shown indicates that is not significant. C ) Model showing the link between TDP-43 and R loop metabolism. Functional TDP43 activity in the nucleus protects genome integrity by preventing R-loop accumulation. However, TDP-43 nuclear dysfunctions results in R loop-dependent DNA damage that eventually might lead to genome instability that could aggravate ALS phenotype.

Article Snippet: SH-SY5Y cells were stably transfected using Fugene HD Transfection Reagent (Promega) with C-terminally GFP-tagged TDP-43 WT vector (PS100010 pCMV6-AC-GFP), C-terminally GFP-tagged TDP-43 A382T mutation vector (CW303334 mutated ORF of RC210639 at nt position 1144, changed from G to A, to obtain A382T, inserted in PS100001) and a C-terminally GFP-tagged TDP-43 G294V mutation vector (CW303287 mutated ORF of RG210639 at nt position 881, changed from G to T, to obtain G294V, inserted in PS100010), purchased from OriGene.

Techniques: MANN-WHITNEY, Two Tailed Test, Flow Cytometry, Fluorescence, Functional Assay, Activity Assay