Fig. 2

Analysis of the YSD plasmids in yeast laboratory strain. (a) Total proteins from CENPK laboratory cells (two independent clones, #1–2), carrying either pGAP-YSD-GFP, pGAP-YSD-yCup1, pGAP-YSD-6xHis plasmids, or the empty vector as control (WT), were subjected to Western blot assay using the anti-FLAG antibody and with anti-GAPDH antibody. Images are representative of three independent experiments. (b) CENPK cells transformed as in (a), were analyzed by immunofluorescence microscopy to visualize the localization of the fusion proteins. For each strain, micrographs are reported of the differential interference contrast (DIC), the immunofluorescence signal (AF555), the nuclear DNA staining (DAPI), and the merged image. Images are representative of three biological replicates for each yeast strain. Scale bar: 5 μm. (c) Growth curves of yeast CENPK cells transformed with either pGAP-YSD-yCup1 (red) or empty (black) plasmids, incubated in SD medium (empty dots), or SD added with 2mM of CuSO₄ (filled dots). Growth rate was monitored for 72 h by OD₆₀₀ measurement (left panel). The OD₆₀₀ final values (72 h) are also reported as graph (right panel). Each column is the mean ± SEM of 4 independent experiments (n = 4, *p < 0,05, ONE WAY Anova with Kruskal-Wallis test was applied for statistical analysis). (d) Living CENPK cells carrying the pYSD-GFP plasmid were directly observed by confocal microscopy. Green fluorescence signal (GFP), differential interference contrast (DIC) micrograph, and merge are reported. Image is representative of three biological replicates; scale bar = 5 μm