Subconfluent primary human foreskin fibroblast cells are infected with rAAV targeting vectors when is being actively transcribed or replicated in proliferative cells

Background reporter gene expression is silenced through density arrest and serum withdrawal. Reporter gene expression is induced by serum stimulation. Single EGFP-Luciferase positive cells are selected using FACS. Cloned cells are expanded and screened for gene targeting events by PCR, Southern blot, and sequencing. FACS histograms are shown for wildtype and rAAV targeted cells following cell cycle entry. FL1-A (EGFP) was plotted against FL2-A (no fluorofor) to center the parent population and allow for selection of the dim EGFP-Luciferase positive cells. The P2 region of the plot indicates the gating used for selection of the EGFP-Luciferase positive portion of the parent population.<p><b>Copyright information:</b></p><p>Taken from "Development of human gene reporter cell lines using rAAV mediated homologous recombination"</p><p></p><p>Biological Procedures Online 2007;9():84-90.</p><p>Published online Jan 2007</p><p>PMCID:PMC2374725.</p><p>Article © by the author(s). This paper is Open Access and is published in Biological Procedures Online under license from the author(s). Copying, printing, redistribution and storage permitted. Journal © 1997-2007 Biological Procedures Online.</p

(A) The indicated Hct116 cells were treated with either aphidicolin (S), nocodazole (G2M), or DMSO (A) for 16 h, and lysates were analyzed by Western blot (γ-tubulin, loading control)

Cyclin E abundance is shown. In each case, >90% of the nocodazole-arrested cells were in G2M and >70% of the aphidicolin-arrested cells were in S phase (not depicted). (B) The indicated Hct116 cell clones were arrested in S phase with a sequential thymidine/hydroxyurea block and released for the indicated time periods. The abundance of cyclin E, phosphorylated cyclin E species, and Grb2 (loading control) are shown. (C) Hct116 cells were treated as in A and lysates were examined for Fbw7 and cyclin E expression. Fbw7-null cell lysates serve as negative control. (D) Hct116 cells were arrested and released from aphidicolin and Fbw7 abundance was determined.<p><b>Copyright information:</b></p><p>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"</p><p></p><p>The Journal of Cell Biology 2008;181(6):913-920.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426948.</p><p></p

(A) Lysates from two independent clones of each Hct116 genotype were analyzed for cyclin E abundance and kinase activity (Grb2, loading control)

(B) Pulse-chase analysis of cyclin E in Hct116 knockout lines. Cells were labeled 1 h after release from aphidicolin arrest (see text). Quantitation of the phosphorimage is depicted in the graph, and the primary data for each isoform-specific cell line is shown. (C) HeLa cells and 293A cells were transfected with the indicated Fbw7 siRNAs (α, β, γ, and CR) or control siRNAs (GFP and C). Cyclin E abundance, kinase activity, and T62 phosphorylation were measured 72 h after transfection. (D) 293A cells and HeLa cells were cotransfected with a FLAG-Fbw7γ expression vector in combination with the indicated siRNAs (γ-tubulin, loading control). (E) 293A cells were transfected with vectors expressing myc-tagged wild-type or P382I cyclin E. Lysates were immunoprecipitated with anti-Myc tag antibody (9E10), and cyclin E abundance, kinase activity, and T62 phosphorylation are shown.<p><b>Copyright information:</b></p><p>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"</p><p></p><p>The Journal of Cell Biology 2008;181(6):913-920.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426948.</p><p></p

(A) Lysates from two independent sets of targeted Hct116 cells were immunoblotted for c-Myc or Grb2 (see for Grb2 panel)

(B) Fbw7 knockout Hct116 cells were treated with cycloheximide as indicated, and lysates were immunoblotted for c-Myc. (C) Real-time PCR analysis of c-Myc mRNA in the indicated Hct116 cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates). (D) Lysates from the indicated Hct116 cells were immunoprecipitated with anti-SREBP1 antibody and immunoblotted for either total SREBP1 (left) or pT426 SREBP1 (right).<p><b>Copyright information:</b></p><p>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"</p><p></p><p>The Journal of Cell Biology 2008;181(6):913-920.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426948.</p><p></p

Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase-4

Ere treated with nocodazole for 16 h. Mitotic cells were shaken off and replated for the indicated times. Cyclin E abundance and activity, as well as cyclin A abundance (a marker of cell cycle synchronization) are shown (*, background band). (B) Hct116 cells or Fbw7-null Hct116 cells were transfected with either control or p21Cip1-specific siRNAs and, after 48 h, treated with aphidicolin (Aph) or nocodazole (Noc). Cyclin E abundance, activity, and S384 phosphorylation are shown. The efficacy of p21 knockdown is shown. (C) Fbw7-null Hct116 cells were treated with aphidicolin or nocodazole and lysates were immunoprecipitated with anti–cyclin E antibody. The amount of CDK2 and Y15–phosphorylated CDK2 bound to cyclin E is shown. (D) Increased Fbw7 binding of cyclin E obtained from mitotic Fbw7-null lysates. Compare the amount of cyclin E that coprecipitates with Fbw7 in lanes 4 and 5 (middle).<p><b>Copyright information:</b></p><p>Taken from "Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase"</p><p></p><p>The Journal of Cell Biology 2008;181(6):913-920.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426948.</p><p></p