Fission yeast Cdc14-like phosphatase Flp1/Clp1 modulates the transcriptional response to oxidative stress [Dataset]

Supplementary Figure S1. Flp1 phosphatase changes its subcellular localization under oxidative stress in a phosphorylation-dependent manner. (a) Live-cell images of gar2-mCherry cells also expressing either Flp1-GFP, Flp1-6A-GFP or Flp1-9A-GFP fusion proteins under unperturbed growth conditions or after treatment with 1mM of H2O2 for 1 h. Asterisks indicate Flp1-GFP nucleoplasmic localization. Scale bar, 5 µm. (b) Line scans of Flp1-GFP and Gar2-mCherry fluorescence of the boxed nuclei from de merged images in (a), represented by a white line and spanning 4 µm from the white closed circle. Fluorescence intensity was normalized to the maximum value after background subtraction. (c) Schematic representation of Flp1 showing all nine RxxS putative phosphorylation sites, which are mutated to alanine in the Flp1.9A-GFP mutant. Asterisks indicate the six serine residues that are mutated to alanine in the Flp1.6A-GFP mutant. (d) The graph shows the percentage of nuclei with Flp1-GFP, Flp1.6A-GFP or Flp1.9A-GFP detected in the nucleoplasm both under unperturbed growth conditions and after treatment with 1mM of H2O2 for 1h. Supplementary Figure S2. flp1+ deletion partially rescues sensitivity of Dsty1 strain to chronic presence of H2O2 in a pap1+-dependent manner. Tenfold serial dilution of indicated strains were spotted onto YES-agar plates without or with 0.6 mM H2O2 and incubated at 30ºC during 72 h. Supplementary Figure S3. (a) Expression of stress-responsive genes indicated in unstressed wildtype and ∆flp1 cells. Total RNA from asynchronous cultures of the indicated strains grown at 30 ºC was extracted and reverse transcribed using a mix of oligo(dT) and random hexamers primers. mRNA levels of ctt1+, gpd1+, hsp9+ and pyp2+ genes were measured by qPCR and normalized with ribosomal 18S mRNA. The amount of mRNAs relative to the wild-type was expressed as means SD in triplicate. Asterisks indicate statistical significance (*, P<0.05; t-test unpaired) versus wild-type. (b) Exponentially growing cultures of strains indicated at 30 ºC were treated with 1mM H2O2 for 1 hour. Total RNA was extracted, separated by electrophoresis and rRNAs stained with methylene blue were used as loading control. Northern-blot analysis was used to measure the expression levels of atf1+ in the indicated strains. Full-length blots are shown in Supplementary Fig. S9. Supplementary Figure S4. Flp1-9A-GFP strain, expressing a nonphosphorylatable flp1 mutant unable to exit the nucleolus under oxidative stress conditions, shows altered the transcriptional profile of genes such as atf1+, pcr1+, ctt1+, gpd1+ and srx1+ induced in response to oxidative stress. mRNA levels of the mentioned genes were measured by qPCR from total RNA extracted from wild-type and Flp1-9A-GFP strains growing in YES medium containing 1mM H2O2. Total RNAs were prepared from treated cells at the time points indicated. mRNA levels were normalized with ribosomal 18S mRNA and determined with respect to time 0´ value, which was considered as 1. Data represent the average of two biological replicates. Supplementary Figure S5. Dflp1 cells show greater resistance to moderate oxidative stress conditions. Asynchronous cultures of indicated strains growing at 30ºC in YES were incubated with 0.2 mM of H2O2 during 1 h. Then, cells were stressed with 2 mM (a) or 25 mM (b) H2O2. Cell viability was measured at the time points indicated by plating the appropriate dilution of cells onto YES agar plates. The number of viable cells was measured after 3 days incubation at 30ºC. Viability is expressed as a percentage of the number of colonies obtained before the addition of the first dose of H2O2. Supplementary Figure S6. Protein levels of Pyp2 phosphatase upon oxidative stress induction. Wild-type and ∆flp1cells expressing a Pyp2-Myc fusion protein were grown in YES medium to midlog phase and treated with 1mM H2O2 for the indicated times. Total extracts were resolved by SDSPAGE and analyzed by Western blot. Pyp2 was detected by incubation with anti-Myc antibody. Tubulin was used as loading control. Normalization of quantified Pyp2 is shown in bar diagrams. Experiment was repeated three times (Supplementary information Fig. S11), and a representative experiment is shown. Blot membranes were cut prior antibodies incubation. Supplementary Figure S7. Viability of Dpcr1 and Dflp1 Dpcr1 mutants in response to oxidative stress. (a) Tenfold serial dilution of indicated strains were spotted onto YES-agar plates without or with 0.6 mM and 1mM H2O2 and incubated at 30ºC during 72 h. (b) Asynchronous cultures of indicated strains growing at 30ºC in YES were incubated with 2 mM of H2O2 during 1 h. Cell viability was measured before and after treatment by plating the appropriate dilution of cells onto YES agar plates. The number of viable cells was measured after 3 days incubation at 30ºC. Viability is expressed as a percentage of the number of colonies obtained before the addition of H2O2 (0´). Data represent the average of two biological replicates. Supplementary Figure S8. Full-length gels and blots relating to Figure 2b,c are shown. Supplementary Figure S9. Full-length gel and blot relating to Figure S3b are shown. Supplementary Figure S10. Unprocessed original scans and full-length immunoblots relating to Figure 5a,b are shown. Supplementary Figure S11. Unprocessed original scans and full-length blots relating to Figure S6 are shown. Supplementary Figure S12. Full-length immunoblots relating to Figure 6a are shown. Supplementary Figure S13. Unprocessed original scans and full-length immunoblots relating to Figure 7 are shown.