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. 2021 Jun 7;11:11919. doi: 10.1038/s41598-021-91480-0

Figure 5.

Figure 5

Pde1/2 positively regulate ATG39 expression via Msn2/4. (A) Effects of PDE2 overexpression on expression of the PATG39-GFP reporter in the msn2 msn4 mutant cells. Wild-type (WT) and msn2 msn4 mutant strains harboring the PATG39-GFP reporter and the plasmids with or without the PDE2 genes were grown at 25 °C until exponential phase and then harvested. The GFP mRNA levels were quantified by qRT-PCR analysis, and relative mRNA levels were calculated using ACT1 mRNA. The values are plotted as the fold change from wild-type cells harboring the empty plasmids. The data show mean ± SEM (n = 4). **P < 0.01 as determined by Student’s t-test. (B) Effects of PDE2 overexpression on expression of mutated PATG39-GFP reporter. Wild-type (WT) cells harboring wild-type or mutated PATG39-GFP reporters and the plasmids with or without the PDE2 genes were grown at 25 °C until exponential phase and then harvested. The GFP mRNA levels were quantified by qRT-PCR analysis, and relative mRNA levels were calculated using ACT1 mRNA. The values are plotted as the fold change from cells harboring wild-type PATG39-GFP reporter and the empty plasmids. The data show mean ± SEM (n = 4). **P < 0.01 as determined by Student’s t-test. (C,D) The ATG39 mRNA levels in pde1 pde2 mutant. Wild-type (WT) and pde1 pde2 mutant strains were grown at 25 °C until exponential phase and treated with 3 μg/ml tunicamycin (TM) (C) or incubated under nitrogen-starved conditions (D) for the indicated time. The ATG39 mRNA levels were quantified by qRT-PCR analysis, and relative mRNA levels were calculated using ACT1 mRNA. The values are plotted as the fold change from wild-type cells at the time of TM addition or nitrogen removal. The data show mean ± SEM (n > 3). *P < 0.05 and **P < 0.01 as determined by Student’s t-test. (E) Cellular localization of Msn2. Wild-type (WT) and pde1 pde2 mutant strains harboring GFP-tagged MSN2 were grown at 25 °C until exponential phase and treated with 3 μg/ml tunicamycin (TM) for 3 h, and subjected to microscopy. The fluorescence intensities were measured, and then the ratios (N/C) of the fluorescence intensity per unit area in the nucleus/that in the cytoplasm were calculated. The graphs show mean ± SEM (n = 30). **P < 0.01 as determined by Student’s t-test. NS, not statistically significant (P > 0.05). Scale bar, 10 μm. (F) Sec63-GFP degradation in ER-stressed pde1 pde2 mutant. Wild-type (WT) and pde1 pde2 mutant strains harboring GFP-tagged SEC63 were grown at 25 °C until exponential phase and treated with 3 μg/ml tunicamycin (TM) for 18 h. Extracts prepared from each cell were immunoblotted with anti-GFP antibodies. The intensities of free GFP were measured and normalized to the Sec63-GFP level. The values are plotted as the fold change from wild-type cells. The data show mean ± SEM (n = 3). *P < 0.01 as determined by Student’s t-test.