HU and DIA induce a UPR-independent response [Dataset]

(A) Canonical ER stress as seen with the luminal ER tag mCherry-AHDL in a wild-type strain after treatment with DTT 2 mM and 5 mM, and with tunicamycin (Tn) 0.6 μg/mL and 2 μg/mL, after a 2-hour incubation. Images are SUM projections of three central Z slices. Scale bars represent 5 microns. (B) Upper panels: Confocal microscopy images of a representative cell of a wild-type strain and an ire1Δ strain, both expressing Cut11-GFP and mCherry-AHDL and showing N-Caps after incubation in 3 mM DIA for 4 hours. Images are SUM projections of three central Z slices. Scale bars represent 5 microns. Lower panels: Quantification of the incidence of the N-Cap phenotype in a wild-type strain and an ire1Δ strain during 3 mM DIA treatment. Graph shows the mean ± SD of three independent repetitions of the experiment, and in each repetition at least 100 cells were accounted for each condition. (C) Viability assay of a wild-type strain and an ire1Δ strain in control conditions, in presence of 2.5 mM DTT and in 3 mM DIA after a 2-day incubation at 30 °C. (D) Principal component analysis separating the three independent repetitions of each of the five conditions addressed in the RNA-Seq. Replicates are very consistent, and HU and DIA conditions are well-differentiated between them and with respect to the control. (E) Heatmap with the expression profile of upregulated and downregulated genes in 3 mM DIA after 30 and 60 min, and in 75 mM HU after 60 and 150 min, with respect to an untreated culture. Three replicates of each condition were assessed. Blue indicates upregulation; yellow indicates downregulation; LFC = Log2 fold change. (F) Volcano plots detailing the significantly upregulated and downregulated genes (LFC ≥ 1 or LFC ≤ −1 respectively, p-value<0.05) in DIA (upper graph) and HU (lower graph), pooling together both times addressed. Red dots show genes that surpass the set thresholds of LFC and p-value. Highlighted genes are some of the common genes in all conditions; ‘variables’ stands for the total number of genes. (G) Upset plots for comparing genes that are upregulated (upper graph) and downregulated (lower graph) between the four treatment conditions. The numbers indicate the genes that are common to the designated combination of conditions. In the case of the unconnected dots, numbers show genes exclusive to that condition alone. Set size graphs indicate the total number of genes in each condition. (H) GO term enrichment for the biological processes shared among the genes common to the four treatment conditions. (I) GO term enrichment for the molecular functions shared among the genes common to the four treatment conditions. Source data for this figure can be found in S1 Data. Raw data from the RNA-Seq analysis is publicly available in Gene Expression Omnibus under the accession number GSE309439.