Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Immunofluorescence staining in monolayer-differentiated NPCs after laser microirradiation. Anti-phospho-histone H2A.X (Ser139) signal serves as a DNA damage marker. Cells were fixed 5-15 minutes after damage generation. b , Live-cell imaging of fluorescently tagged PHF14 complex proteins and PARP1 and ZMYND8 as positive controls (transient transfection into U2OS cells). DNA damage was induced with 355 nm laser at the indicated location. c , Quantified recruitment kinetics from live-cell laser microirradiation experiments for the PHF14 complex and controls (in U2OS for RAI1, PARP1 and ZMYND8, other proteins from HEK293T cells, no difference was observed between different cell lines in terms of recruitment kinetics). Time is set to 0 at the time of DNA damage induction and intensities are normalized such that the maximum reached is 1. A first-order exponential equation f(t) = 1-exp(−t / τ) was fitted to all data per protein. The time required for the normalized fluorescence intensity to reach 50% of its maximal value is represented as t50. The last panel includes fitted curves from the other panels only. n = 8 (PHF14), 14 (HMG20A), 8 (TCF20), 8 (RAI1), 8 (PARP1), 6 (ZMYND8). d , Laser microirradiation in transiently transfected U2OS cells after treatment with 1 μM olaparib for 1 h or equivalent amount of DMSO. All scale bars = 10 μm.

Article Snippet: Lentivirus was produced in HEK293T cells using a 2 nd generation lentiviral packaging system (psPAX2 – Addgene plasmid #12260 and pMD2.G – Addgene #12259 from Didier Trono) and pRRL.EF1a.HA.NLS.Sce(opt).T2A.BFP (Addgene #32628 from Andrew Scharenberg) and CalPhos transfection kit according to manufacturer’s instructions (Takara).

Techniques: Immunofluorescence, Staining, Marker, Live Cell Imaging, Transfection, Fluorescence

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Immunofluorescence staining in monolayer-differentiated NPCs after laser microirradiation. Anti-phospho-histone H2A.X (Ser139) signal serves as a DNA damage marker. Cells were fixed 1 h after DNA damage was generated. Laser microirradiation in transiently transfected U2OS cells, 1 hour after DNA damage induction. b , Live-cell imaging of mEGFP-HMG20A and mRuby-PHF14 transiently transfected into U2OS cells. 1 hour after damage induction. c , Live-cell imaging of mEGFP and TCF20-mCherry (transient co-transfection into U2OS cells). DNA damage was induced with 355 nm laser at the indicated location. d , Immunofluorescence staining in control and Phf14 KO ESCs after laser microirradiation. e , Live-cell imaging of mEGFP-HMG20A, transiently transfected into control and Phf14 KO ESCs. DNA damage was induced with 355 nm laser at the indicated location. f , Quantified recruitment kinetics for mEGFP-HMG20A in control and Phf14 KO ESCs. g , Immunofluorescence staining in U2OS cells after laser microirradiation. DNA damage was induced after treatment with 1 μM olaparib for 1 h or equivalent amount of DMSO. All scale bars = 10 μm.

Article Snippet: Lentivirus was produced in HEK293T cells using a 2 nd generation lentiviral packaging system (psPAX2 – Addgene plasmid #12260 and pMD2.G – Addgene #12259 from Didier Trono) and pRRL.EF1a.HA.NLS.Sce(opt).T2A.BFP (Addgene #32628 from Andrew Scharenberg) and CalPhos transfection kit according to manufacturer’s instructions (Takara).

Techniques: Immunofluorescence, Staining, Marker, Generated, Transfection, Live Cell Imaging, Cotransfection, Control

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Confocal microscopy images of Cy5-labeled dsDNA in phase separated protein droplets showing droplets merging (indicated by the white arrow). Scale bar = 10 μm. b , Whole-droplet and partial FRAP of the phase separated droplets for Cy5-labeled DNA. Scale bars = 5 μm. c , Phase contrast images of condensates that are formed with indicated proteins with and without 1,6-hexanediol. 300 nM of proteins were used. Images were taken with a widefield Nikon Ti-E microscope. Scale bar = 20 μm. d , Schematic of TCF20 protein. NLS1 (aa 1254-1268) precedes the Gln1269* patient mutation, explaining the nuclear localization of the TCF20(1-1268) construct. e , TCF20(1-1268)-mEGFP transfected into U2OS cells. White outlines indicate nuclear borders. Scale bar = 10 μm. f , Correlation between fluorescent signal intensity in TCF20(1-1268)-mEGFP transfections with the formation of foci. Data from 3 frames, normalized within frame with the average signal from cells without foci set to 1. g , TCF20(1-1268)-mEGFP before and immediately after the addition of 1,6-hexanediol and digitonin to final concentrations of 5% and 5 μg/mL, respectively. Imaged with 5 s frame interval. Scale bar = 10 μm. h , Confocal microscope images of full length TCF20-mEGFP and TCF20(1-1268)-mEGFP accumulation at DNA damage sites after damage induction with 355 nm laser. Scale bars = 10 μm.

Article Snippet: Lentivirus was produced in HEK293T cells using a 2 nd generation lentiviral packaging system (psPAX2 – Addgene plasmid #12260 and pMD2.G – Addgene #12259 from Didier Trono) and pRRL.EF1a.HA.NLS.Sce(opt).T2A.BFP (Addgene #32628 from Andrew Scharenberg) and CalPhos transfection kit according to manufacturer’s instructions (Takara).

Techniques: Confocal Microscopy, Labeling, Microscopy, Mutagenesis, Construct, Transfection

Journal: Scientific Reports

Article Title: MicroRNA regulation of CYP 1A2, CYP3A4 and CYP2E1 expression in acetaminophen toxicity

doi: 10.1038/s41598-017-11811-y

Figure Lengend Snippet: Transfection of miRNA mimic and inhibitor into HepaRG cells and its effect on the protein levels in APAP treated cells (N = 3). Western blot analysis of miR-122 mimic and inhibitor transfected cells treated with APAP for 12 h. ( A ) CYP1A2 and ( B ) CYP3A4: Ctrl = Control untreated; APAP = 20 mM; miR-122 Mimic 5 nM; miR-122 Inhibitor 100 nM. Uncropped image of blots for CYP1A2 and CYP3A4 are shown in Figure . Western blot analysis of miR-378 mimic and inhibitor transfected cells ( C ) CYP2E1: Ctrl = Control untreated; APAP = 20 mM; miR-378a Mimic 5 nM; miR-378a Inhibitor 50 nM. Uncropped image of blot for CYP2E1 shown in Figure . Data are normalized to β-Actin (*p < 0.05, **p < 0.01) and error bars represent standard error of the mean (SEM). ( D ) ALT levels in miR-122 and miR-378a mimic and inhibitor transfected cells treated with 20 mM APAP for 12 h. “h” denotes hour.

Article Snippet: HEK293T cells were seeded in 96-well plates (3 × 10 4 cells/well) for 24 h. HEK293T cells were co-transfected in OPTI MEM media (ThermoFisher Scientific, Carlsbad, CA) with 200 ng of 3′ UTR luciferase reporter plasmids and miR-122 mimic, miR-122 inhibitor or negative control plasmid pEZX-MT06 (GeneCopoeia, Rockville, MD) with Attractene transfection reagent (Qiagen, Valencia, CA).

Techniques: Transfection, Western Blot