NE deformation and Spt23/Mga2 activation are independent of Sir4. (A) Representative images of wild-type and sir4Δ cells expressing the ER marker Sec63^GFP from a centromeric plasmid. Cells were imaged using live fluorescence microscopy after growth in SD-Leu+Ade and 90 min in the presence of edelfosine or control. Quantification of non-round nuclei is shown beside the microscopy images. (B and C) Representative images of ^GFPSpt23 (B) or ^GFPMga2 (C) expressed from a centromeric plasmid under the constitutive GPD promoter in wild-type or sir4Δ cells imaged after growth in SD-Leu+Ade and 30 min in the presence of edelfosine or the vehicle. Quantification of nucleoplasmic signal is shown beside microscopy images. For all microscopy quantifications (A, B, and C), circles represent the percentage of cells displaying the indicated phenotype in each experiment (n = 100 cells per treatment), while the bar represents the mean of all independent experiments ± SD. Differences between wild-type and sir4∆ were found to be non-significant in both control and edelfosine treatments as determined by two-way ANOVA with Šídák’s multiple comparisons test (N = 4 for NE, N = 3 for Spt23 and Mga2). (D) qPCR of Spt23 and Opi1 targets in wild-type and sir4Δ cells treated for 60 min with edelfosine or vehicle expressed as ln(EDLF/Ctrl). Bars represent mean ± SD for three independent experiments while circles represent individual experiments. * indicates P value <0.05 as determined by unpaired t tests with Holm-Šídák correction for multiple comparisons. For all panels: Ctrl = vehicle; EDLF = edelfosine; scale bars represent 2 µm.