Journal: Cell Discovery

Article Title: A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A

doi: 10.1038/celldisc.2016.14

Figure Lengend Snippet: Acetylation of lysine 150 on Cdc25A negatively regulates the G2/M checkpoint in response to IR. ( a ) Knockout of endogenous Cdc25A in HeLa cells through CRISPR-Cas9. ( b ) Re-expression of wild-type (WT) or K150R mutant Cdc25A in the Cdc25A knockout cell line through pSIN lentivirus. ( c, d ) Degradation rate of WT-Cdc25A and K150R-Cdc25A mutant post IR (6 Gy) using western blot. ( e, f ) Cdc25A KO HeLa cells stably re-expressing WT-Cdc25A or K150R-Cdc25A mutant were exposed to IR (6 Gy). The cells were harvested and analyzed using flow cytometry after incubation with 100 ng ml −1 of nocodazole for 10 h. The percentage of cells positive for phospho-histone H3 (p-H3) is indicated. N =3. Bars indicate the s.e.m. *** P <0.001, Student’s t- test.

Article Snippet: After the cells were centrifuged, the cell pellet was suspended in 100 ml of PBS containing 1% bovine serum albumin and 0.75 μg of a polyclonal antibody that specifically recognizes the phosphorylated form of histone H3 (Upstate Biotechnology, Lake Placid, NY, USA), and incubated for 1.5 h at room temperature.

Techniques: Knock-Out, CRISPR, Expressing, Mutagenesis, Western Blot, Stable Transfection, Flow Cytometry, Incubation

Journal:

Article Title: Cytoplasmic Localization of Human cdc25C during Interphase Requires an Intact 14-3-3 Binding Site

doi:

Figure Lengend Snippet: Cellular localization of cdc25C in human cells. (A) Indirect immunofluorescence with the cdc25C antibodies was performed in multiple cell types. U-2OS cells immunostained with either TC14 (left) or TC113 (right) are shown in the top panels. The primary human fibroblasts, MRC-5 (bottom left) and WI-38 (bottom right), were immunostained with antibody TC113. In each pair, the left panel shows antibody staining while the right panel shows the DAPI stain of the same field (original magnification, ×100). (B) MRC-5 cells were immunostained with polyclonal sera specific for phospho-histone H3 (α pH3) (17), a MAb to cdc25C (TC113), and DAPI, as indicated, and visualized by confocal microscopy. The merge of the cdc25C and the pH3 staining is shown in the bottom left panel. A cell that stains positively for histone H3 and shows partial chromatin condensation (DAPI) is indicated by the thick arrow. A cell that does not stain with the phospho-histone H3 antibody in which the chromatin is not condensed is indicated by the thin arrow (original magnification, ×63). (C) U-2OS cells were treated with mimosine to induce a G1 arrest. Six hours after mimosine release, cells were γ irradiated (γ-IR) with a dose of 6 Gy and then incubated in either the presence or absence of the crm1 inhibitor leptomycin B (LMB) for an additional 18 h. Cells were immunostained with either an anti-cdc25C antibody (TC113) or an anti-cyclin B1 antibody (CB169). In each pair, the left panel shows immunofluorescence while the right panel shows the DAPI stain of the same field (original magnification, ×100). The cell cycle profiles of the cells in mimosine (mimosine), cells 6 h after release from mimosine (6 hours), and the γ-irradiated cells (γ-IR) and irradiated cells treated with leptomycin B (γ-IR + LMB) are shown.

Article Snippet: The phosphorylated histone (phospho-histone) H3-specific antiserum (Upstate Biotechnology) was used at a dilution of 1:200 for immunofluorescence.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Irradiation, Incubation