Journal: Cell Reports Methods

Article Title: Data-driven microscopy allows for automated context-specific acquisition of high-fidelity image data

doi: 10.1016/j.crmeth.2023.100419

Figure Lengend Snippet: Key resources table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemical, peptides and recombinant proteins Hoechst Themo Scientific Cat#62249 HEPES Gibco Cat#15630056 Phosphate buffered saline (PBS) Gibco Cat#20012068 Bovine Serum Albumin VWR Cat#422351S Fibronectin Sigma Aldrich Cat# F0895 Paraformaldehyde (PFA) Themo Scientific Cat#28908 Ampicillin Sigma Aldrich Cat#A9518 Kanamycin Sigma Aldrich Cat#K1377 Dulbecco's Modified Eagle's Media (DMEM) Gibco Cat#61965-059 Roswell Park Memorial Institute (RPMI 1640) Gibco Cat#61870044 Fetal Bovine Serum (FBS) Gibco Cat#10270106 Luria-Bertani's (LB) Media Sigma Aldrich Cat#L3022 Critical commericial assays QIAGEN Plasmid Mini Kit QIAGEN Cat#12143/12145 Transfection kit Invitrogen Cat#L3000008 Experimental models: Cell lines HeLa WT Sigma Aldrich Cat#93021013-1VL; RRID: CVCL_0030 H1299 WT Shafqat-Abbasi et al. 44 N/A H1299 WT - mKate-Paxillin Shafqat-Abbasi et al. 44 N/A Recombinant DNA Plasmid: pmScarlet_alphaTubulin_C1 Addgene Cat#85045; RRID: Addgene_85045 Plasmid: pcDNA3.1 LifeAct E2-Crimson This paper N/A Plasmid: pEF1 ITGB1 wt Timothy Springer Lab N/A Plasmid: ITGA5-GFP Addgene Cat#15238; RRID: Addgene_15238 Plasmid: GFP plasmid This paper N/A Bacterial strains Yersinia Psuedotuberculosis WT Maria Fällman Lab N/A Software and algorithms Julia https://julialang.org/ N/A Nim programming language https://nim-lang.org/ N/A DDMFramework.jl https://github.com/nordenfeltLab/DDMFramework.jl https://doi.org/10.5281/zenodo.7590417 DDMServer.jl https://github.com/nordenfeltLab/DDMServer.jl https://doi.org/10.5281/zenodo.7670863 DDMTransfection.jl https://github.com/nordenfeltLab/DDMTransfection.jl https://doi.org/10.5281/zenodo.7590424 DDMInfection.jl https://github.com/nordenfeltLab/DDMInfection.jl https://doi.org/10.5281/zenodo.7671048 DDMMigration.jl https://github.com/nordenfeltLab/DDMMigration.jl https://doi.org/10.5281/zenodo.7590361 Microscope CLI https://github.com/nordenfeltLab/ddm_microscope_cli https://doi.org/10.5281/zenodo.7670892 Other μ-Slide 8-Well Glass Bottom slides Ibidi Cat#80821 μ-Slide 4-Well Ph+ Glass Bottom slides Ibidi Cat#80447 Open in a separate window Key resources table .

Techniques: Recombinant, Saline, Modification, Plasmid Preparation, Transfection, Software, Microscopy

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: E2- and E1-dependent transient DNA replication. C33A cells were transfected with HPV-31 E1 and HPV-31 Y138 mutants, along with pFLORI31 and pRL constructs. After 72 h, cells were lysed, and firefly and Renilla luciferase levels were measured using Dual-Glo luciferase reagent. Firefly luciferase levels were normalized to Renilla luciferase levels. Values are expressed as means ± SEM (n = 8). (A) P < 0.0001 by 1-way analysis of variance (ANOVA); #, P = 0.045 by two-tailed t test; *, P < 0.0001 by two-tailed t test. (B) Coexpression of constitutive kinase-active FGFR3 K650E. FGFR3 K650E suppressed WT (fold change < 1) but not Y138F replication (fold change = 1). Values are expressed as means ± SEM (n = 8). *, P < 0.01 by two-tailed t test comparing FGFR3 K650E to controls. (C) Coexpression of FGFR2 in 0.5% fetal bovine serum (FBS). Values are expressed as means ± SEM (n = 8). * P < 0.001 by two-tailed t test compared to pCI control groups.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection, Construct, Luciferase, Two Tailed Test

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: (A) HEK293TT cells were transfected with FLAG-FGFR3 K650E and FLAG-HPV-31 E2 mutants. Cells were lysed, and FLAG-FGFR3 K650E immunoprecipitated (IP) with FGFR3 antibodies. Complexes were blotted with FLAG antibodies. (B) HEK293TT cells were transfected with FLAG-FGFR3 K650E and FLAG-HPV-31 E2 mutants. Cells were lysed, and HPV-31 E2 immunoprecipitated with FLAG gel. Complexes were blotted with PY-1000 and FLAG antibodies. Densitometry from three independent experiments is shown. Phosphotyrosine E2 levels were normalized to total E2 levels.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: (A) Illustration of E2 truncations and deletions. HEK293TT cells were transfected with FLAG-FGFR3 K650E and these FLAG-HPV-31 E2 constructs. (B) After 48 h, cells expressing aa 107 to 372 and aa 176 to 372 were treated with 20 μM MG132 for 6 h. Cells were lysed, and FLAG-FGFR3 K650E immunoprecipitated with FGFR3 antibodies. Complexes were blotted with FLAG antibodies. (C) FLAG-HPV-31 E2 and FLAG-FGFR3 K650E immunoprecipitated with FLAG antibodies. Complexes were blotted with PY-1000 and FLAG antibodies (D) FLAG-FGFR3 K650E was immunoprecipitated with FGFR3 antibodies. Complexes were blotted with FLAG antibodies. (E) FLAG-HPV-31 E2 and FLAG-FGFR3 K650E immunoprecipitated with FLAG antibodies. Complexes were blotted with PY-1000 and FLAG antibodies.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection, Construct, Expressing, Immunoprecipitation

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: E2 Y138 mutants are nuclear, but Y138E do not form replication foci. CV-1 cells were transfected with FLAG-HPV-31 E2 Y138 mutants, HA-HPV-31 E1, and HPV-31 ori. Immunofluorescence staining was carried out with FLAG (red) and E1 (green) antibodies at 100×; 4′,6-diamidino-2-phenylindole (DAPI; blue) was used to stain the nuclei.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: (A) HeLa cells were transfected with FLAG-WT and E2 Y138 mutants. After 48 h, ChIP assays were performed using the ChIP-IT express chromatin immunoprecipitation kit (Active Motif) with FLAG antibodies. HPV-18 LCR DNA was amplified using RT-PCR, and values were normalized to those of input DNA. Control = no FLAG E2 DNA; WT = 1. Values are expressed as means ± SEM. *, P ≤ 0.05 by two-tailed t test compared to control. (B) HeLa cells were transfected with FLAG-HPV-31 E1 and FLAG-WT or Y138 E2 mutants. After 48 h, the ChIP assays were performed with rat anti-E1 antibodies. HPV-18 LCR DNA was amplified using RT-PCR, and values were normalized to those of input DNA. No E1 = 1. Values are expressed as means ± SEM. *, P ≤ 0.05 by two-tailed t test compared to no E1 group. No statistically significant difference was found between WT and Y138 mutant groups. E1 only (n = 6), no E1 (n = 12), E1+ WT (n = 11), E1 + Y138F (n = 12), and E1 + Y138E (n = 12). (C) HeLa cells were transfected with WT and Y138 E2 mutants. After 48 h, RNA was isolated and cDNA was synthesized. Real-time PCR was used to measure HPV-18 E6 cDNA and normalized to actin cDNA.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection, Chromatin Immunoprecipitation, Amplification, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Mutagenesis, Isolation, Synthesized, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: Y138E binds HPV-31 E1 and GPS-2. (A) HEK293TT cells were transfected with HA-HPV-31 E1 and FLAG-HPV-31 E2 Y138 mutants. FLAG pulldown was completed with M2 gel and immunoblotted with 16E1 and FLAG antibodies. Densitometry for four independent experiments is shown. E1 levels were normalized to E2 levels. (B) HEK293TT cells were transfected with HA-GPS2 and FLAG-HPV-31 E2 Y138 mutants. FLAG pulldown was completed with M2 gel and immunoblotted with GPS-2, HPV-16 E1, and FLAG antibodies.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: Y138E does not bind to the Brd4 C-terminal domain (CTD). (A) HEK293TT cells were transfected with FLAG-Brd4 full-length and FLAG-HPV-31 E2 Y138 mutants. Brd4 pulldown with Brd4 antibodies and immunoblotted with Brd4 and FLAG antibodies. (B) HEK293TT cells were transfected with glutathione S-transferase (GST)-CTD and FLAG-HPV-31 E2 Y138 mutants. FLAG pulldown with M2 gel and immunoblotted with GST and FLAG antibodies. (C) HEK293TT cells were transfected with GST-CTD and FLAG-HPV-31 E2 Y138 mutants. GST pulldown with glutathione Sepharose beads and immunoblotted with GST and FLAG antibodies. (D) HEK293TT cells were transfected with GST-BID and FLAG-HPV-31 E2 Y138 mutants. GST pulldown with glutathione Sepharose beads and immunoblotted with GST and FLAG antibodies.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: The Brd4 CTD increased binding to CDK9 in the presence of Y138E. (A) HEK293TT cells were transfected with GST-CTD and FLAG-HPV-31 E2 Y138 mutants. GST pull down with glutathione Sepharose beads and immunoblotted with GST, FLAG, and CDK9 antibodies. (B) Western blot images were quantified for CDK9 and Brd4 CTD expression. Values are expressed as means ± SEM, n = 3. CDK9 expression was normalized to Brd4 CTD expression for coimmunoprecipitation, WT = 1.

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Binding Assay, Transfection, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

doi: 10.1128/JVI.01801-18

Figure Lengend Snippet: FIT-039 impairs HPV-31 transcription but does not rescue Y138E replication. (A) C33A cells were transfected with control plasmid or HPV-31 E2 and the pGL2-E2BS-Luc reporter plasmid. After 6 h, 5 or 10 μM FIT-039 was added to the cells. After 42 h, cells were lysed and firefly luciferase levels were measured. Values are expressed as fold change compared to no E2. Values are expressed as means ± SEM (n = 8). P < 0.0001 by 1-way ANOVA. *, P < 0.0001 by two-tailed t test compared to dimethyl sulfoxide (DMSO). (B) C33A cells were transfected with HPV-31 E1 and HPV-31 Y138 mutants, along with pFLORI31 and pRL constructs. After 6 h, 5 μM FIT-039 was added, and after an additional 42 h, cells were harvested and firefly and Renilla luciferase levels measured. Firefly luciferase levels were normalized to Renilla luciferase levels. Values are expressed as means ± SEM (n = 8).

Article Snippet: CV-1 cells were transfected onto coverslips with 1 μg pCDNA3-FLAG-HPV-31 E2 constructs using lipofectamine (Life Technologies).

Techniques: Transfection, Plasmid Preparation, Luciferase, Two Tailed Test, Construct