25 research outputs found
Overexpression of <i>RECQL4</i> results in increased RAD51 foci and decreased tail moment.
No full text(A) Overexpression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. U2OS cells were transfected with an empty plasmid or a plasmid expressing RECQL4 or RECQL4-K508A under a CMV promoter. The cells were either mock or cisplatin treated for one hour and after a two-hour recovery, imaged for RAD51 foci or DAPI by immunofluorescence. RAD51 foci was quantified from 200 cells per condition for each experiment. The experiment was performed three to five times and the median was graphed (Unprocessed foci count in S9 Data). Representative images are shown. (B) Overexpression of RECQL4 results decreased tail moment following cisplatin exposure, which is dependent on its helicase activity. U2OS cells were treated similarly to the immunofluorescence experiment, before being harvested for neutral comet assay. At least 40 comets were counted per condition for each experiment. The experiment was performed four times and the mean and standard deviation was graphed (Unprocessed tail moments in S10 Data).</p
Hrq1 function and regulation in ICL repair is distinct from intrastrand crosslink repair.
No full text(A) Hrq1 functions in a different pathway as Rev1 and Ubc13 to repair ICL. The indicated yeast strains were five-fold serial diluted onto YPD medium containing DMSO and/or YPD medium containing the indicated amount of MMC. The plates were photographed after 2 days of incubation at 30°C in the dark. (B) Hrq1 protein levels does not decreased upon 100 μg/ml MMC treatment. Exponentially growing cells with Hrq1-9xMYC were incubated with cycloheximide in the presence or absence of 100 μg/ml MMC and/or 0.1% DMSO. Protein extracts were analyzed by western blot for Hrq1 protein levels (α-MYC) or a loading control, Kar2 (α-Kar2) at the indicated time points. Quantification from three separate experiment is shown, with mean and SEM graphed (Raw densitometry data in Sheets S-U in S1 Data). (C) Hrq1 functions in the same pathway as Rad5 and Ubc13 to repair intrastrand adducts. The indicated yeast strains were five-fold serial diluted onto YPD medium before being treated with the indicated dosages of UV-C. The plates were photographed after 2 days of incubation at 30°C in the dark. (D) Hrq1 functions in different pathway as Rev1 to repair intrastrand adducts. The indicated yeast strains were five-fold serial diluted onto YPD medium before being treated with the indicated dosage of UV-C. The plates were photographed after 2 days of incubation at 30°C in the dark. (E) Hrq1 protein levels decreased following UV-C treatment. Exponentially growing Hrq1-9xMYC cells were incubated with cycloheximide then treated with indicated dosage of UV-C. Protein was extracted similarly to (B). Quantification is from three separate experiments with mean and SEM graphed (Raw densitometry data in Sheets V-X in S1 Data). (TIF)</p
Hrq1 protein levels are stabilized in the absence of the E3 Ub-ligase, <i>RAD16</i>.
No full text(A) Deletion of Rad16 stabilizes Hrq1 following cisplatin exposure. Hrq1-9xMYC expressing wild-type (WT) or rad16Δ, cells were incubated with cycloheximide in the presence or absence of 100 μg/ml cisplatin and/or 0.1% DMSO. Note blot from Fig 1C was reshown for comparison. (B) Quantification of the proportion of Hrq1 remaining relative to time 0 (before CHX addition) and the loading control, Kar2, are plotted on the graph in log scale from WT and rad16Δ cells. Each experiment was performed three to 5 times with standard error plotted (Raw densitometry data in Sheets F-H in S1 Data). Note that the WT cisplatin treated time course is replotted from Fig 1C, for direct comparison to rad16Δ cisplatin treated cells.</p
Although Hrq1 is needed for cisplatin resistance, it is degraded by the proteasome upon cisplatin exposure.
No full text(A) HRQ1-null cells are sensitive to cisplatin. Wild-type (WT) or hrq1Δ disrupted cells were five-fold serially diluted on medium containing 30 μg/ml cisplatin and/or 0.1% DMSO, grown for 48 hours at 30°C, and photographed. (B) Hrq1 level is stable following treatment with other DNA damaging agents: cisplatin (100 ug/ml), MMS (0.03%), IR (100 Gy), HU (100 mM). Exponentially growing cells with Hrq1-9xMYC were treated with the indicated drugs for 2 hours before being harvested for western. (C) Hrq1 protein levels are decreased upon cisplatin treatment. Exponentially growing cells with Hrq1-9xMYC were incubated with cycloheximide in the presence or absence of 100 μg/ml cisplatin and/or 0.1% DMSO. Quantification of the proportion of Hrq1 remaining relative to time 0 (before CHX addition) and the loading control, Kar2. The experiment was performed five times with mean and standard error plotted (Raw densitometry data in Sheets A-E in S1 Data). It is important to note that Hrq1 and the loading control, Kar2, were analyzed from the same gels to account for pipetting errors. Since Hrq1 is not as abundant as the loading control, there is a limitation for the densitometry analysis. (D) The proteasome degrades Hrq1 following cisplatin exposure. PDR5 disrupted cells were untreated (0.1% DMSO), cisplatin treated, or pretreated for one hour with 50 μM MG-132 before cisplatin addition with 0.1% DMSO. Cycloheximide chases were performed the similarly as (B) but further timepoints were taken.</p
