Although Hrq1 is needed for cisplatin resistance, it is degraded by the proteasome upon cisplatin exposure.

(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.