Journal: bioRxiv

Article Title: TORC1 reactivation by pheromone signaling revealed by phosphoproteomics of fission yeast sexual reproduction

doi: 10.1101/2024.06.04.597361

Figure Lengend Snippet: A. Western blot of Rps6 phosphorylation in WT and cycΔ5 fus1 opto cells kept in the dark. Cells were either grown in MSL + ammonium sulfate (+ NH4), MSL + Glutamate (+Glu) or starved for 6h30 (WT) or 3h ( cycΔ5 fus1 opto ) without nitrogen (-N). Homothallic strains ( h90 ) or mix of mating types ( h+/h- ) mate in these conditions. The rps601Δ rps602 AA strain was used as control for the specificity of the phospho-specific antibody. Rps6 total levels are shown at the bottom. B. Phospho-Rps6, phospho-Psk1 and phospho-Gad8 levels in WT heterothallic ( h- ) and homothallic ( h90 ) cells starved for 6h15 and then treated with either rapamycin (300 ng/ml), Torin1 (5 µM) or DMSO for 30 min. See for further Torin1 treatment. C. Phospho-Rps6 and phospho-Psk1 in WT and tor2 S1837E mutant treated with rapamycin or DMSO as in (B). D. Phospho-Rps6 levels in wild-type and psk1Δ mutant after 6h30 starvation. E. Time course of phospho-Rps6 and phospho-Psk1 in h-sxa2Δ cells starved in the presence of P-factor (1µg/ml) or MetOH. Starvation and P-factor addition occurred at T = 0 min.

Article Snippet: For experiment with Torin1 and Rapamycin ( and ), strains were incubated in MSL-N for 6h15 and 40 ml of cells were then treated with either 40 µl of Torin1 (5 mM stock in DMSO; final concentration 5 µM) (LC Laboratories; T-7887), 40 µl of Rapamycin (300 µg/ml stock in DMSO; final concentration 300 ng/ml) (LC Laboratories; R-5000) or 40 µl of DMSO and incubated for an additional 30 min at 30°C.

Techniques: Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: TORC1 reactivation by pheromone signaling revealed by phosphoproteomics of fission yeast sexual reproduction

doi: 10.1101/2024.06.04.597361

Figure Lengend Snippet: A. Phospho-Rps6 and phospho-Psk1 levels in WT heterothallic ( h- ) and homothallic ( h90 ) cells starved for 6h15 and then treated with Torin1 (5 or 25 µM) or DMSO (0) for 30 min. B. CFP-Atg8 cleavage in WT and atg1Δ mutants after 4h and 8h of nitrogen starvation in MSL. The cells were pre-grown in MSL + glutamate (top) or MSL + ammonium (bottom). C. Time course of phospho-Psk1 in h-sxa2Δ atg18aΔ cells transferred to MSL + 0.75 mg/ml glutamate in the presence of P-factor (1µg/ml) or MetOH at T = 0 min.

Article Snippet: For experiment with Torin1 and Rapamycin ( and ), strains were incubated in MSL-N for 6h15 and 40 ml of cells were then treated with either 40 µl of Torin1 (5 mM stock in DMSO; final concentration 5 µM) (LC Laboratories; T-7887), 40 µl of Rapamycin (300 µg/ml stock in DMSO; final concentration 300 ng/ml) (LC Laboratories; R-5000) or 40 µl of DMSO and incubated for an additional 30 min at 30°C.

Techniques:

Journal: bioRxiv

Article Title: TORC1 reactivation by pheromone signaling revealed by phosphoproteomics of fission yeast sexual reproduction

doi: 10.1101/2024.06.04.597361

Figure Lengend Snippet: A. Western blot of Rps6 phosphorylation in WT and cycΔ5 fus1 opto cells kept in the dark. Cells were either grown in MSL + ammonium sulfate (+ NH4), MSL + Glutamate (+Glu) or starved for 6h30 (WT) or 3h ( cycΔ5 fus1 opto ) without nitrogen (-N). Homothallic strains ( h90 ) or mix of mating types ( h+/h- ) mate in these conditions. The rps601Δ rps602 AA strain was used as control for the specificity of the phospho-specific antibody. Rps6 total levels are shown at the bottom. B. Phospho-Rps6, phospho-Psk1 and phospho-Gad8 levels in WT heterothallic ( h- ) and homothallic ( h90 ) cells starved for 6h15 and then treated with either rapamycin (300 ng/ml), Torin1 (5 µM) or DMSO for 30 min. See for further Torin1 treatment. C. Phospho-Rps6 and phospho-Psk1 in WT and tor2 S1837E mutant treated with rapamycin or DMSO as in (B). D. Phospho-Rps6 levels in wild-type and psk1Δ mutant after 6h30 starvation. E. Time course of phospho-Rps6 and phospho-Psk1 in h-sxa2Δ cells starved in the presence of P-factor (1µg/ml) or MetOH. Starvation and P-factor addition occurred at T = 0 min.

Article Snippet: For experiment with Torin1 and Rapamycin ( and ), strains were incubated in MSL-N for 6h15 and 40 ml of cells were then treated with either 40 µl of Torin1 (5 mM stock in DMSO; final concentration 5 µM) (LC Laboratories; T-7887), 40 µl of Rapamycin (300 µg/ml stock in DMSO; final concentration 300 ng/ml) (LC Laboratories; R-5000) or 40 µl of DMSO and incubated for an additional 30 min at 30°C.

Techniques: Western Blot, Mutagenesis