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. 2015 Mar 24;10(3):e0121059. doi: 10.1371/journal.pone.0121059

Fig 7. High levels of ROS accompany the death of mini_cnx1 cells under nitrogen starvation and chronological aging.

Fig 7

Accumulation of ROS in cells under nitrogen starvation (A) and (B) during aging cultures (CLS), was determined using dihydroethidium (DHE) staining. Calnexin mutants mini_cnx1 (mini, SP18344), lumenal_cnx1 (lum,SP18346), lumenalTM-cnx1 (lum-tm, SP18348), Δhcd_cnx1 (hcd, SP18350), mini_cnx1+lumenalTM-cnx1 (lum-tm + mini, SP18282) were evaluated in comparison to WT calnexin (WT,SP18342), and to the vacuolar-proteases deficient strain isp6psp3 (SP18417). (A) Cells were grown in minimal media (EMM) until exponential phase (0.3–0.4 OD/ml), washed twice in water, and then starved for nitrogen in EMM minus nitrogen (EMM-N). Culture samples were taken at exponential phase (EP) and days 1, 4 and 6 of nitrogen-starvation, stained with DHE (10 μM) for 30 min at 30°C. Sample of 10000 cells were analyzed by flow cytometry (BD FACSAria). (B) Cells were grown in CLS medium [EMM supplemented with all amino acids at 112 mg/L (EMMC)]. Culture samples were taken at EP and days 1, 4 and 7 of stationary phase and processed as above for DHE staining. For each-bar graph, data represent means ± s.e.m. of DHE-positive cells per viable cells (as determined by Phloxin B staining, see M&M). Each experiment was repeated at least three times.