Additional file 3 of TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae
Creators: name: Alfatah, Mohammad
name: Wong, Jin Huei
name: Vidhya Gomathi Krishnan
name: Lee, Yong Cheow
name: Sin, Quan Feng
name: Goh, Corinna Jie Hui
name: Kiat Whye Kong
name: Lee, Wei Ting
name: Lewis, Jacqueline
name: Hoon, Shawn
name: Arumugam, Prakash
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Additional file 3: Fig. S4. TORC1 is required to maintain the expression of TGC genes in glucose-containing growth medium. Wild type cells grown into mid-log phase in YPD medium were treated with either DMSO or rapamycin (200 nM). Aliquots of the cultures were taken after 0, 0.5, 1 and 2 hours. RNA was extracted from the cultures and the expression of the TGC genes was analyzed by Real-Time qRT-PCR. Data are presented as means ± standard deviation (n = 2 replicates). Fig. S5. TORC1 regulates the glucose-responsive genes independently of Bcy1 T129 dephosphorylation. Cells expressing wild type Bcy1 and mutant bcy1- T129D were grown to logarithmic phase in SC-EG medium. Glucose was added to the cultures at the final concentration of 2% along with either rapamycin (200 nM) or DMSO. Aliquots of the cultures were taken after 0’, 15’ and 30’. RNA was extracted from the cultures and the expression of the glucose response genes DHR2, CIT1 and RME1 were analyzed by Real-Time qRT-PCR. Data are presented as means ± standard deviation (n = 2 replicates). Fig. S6. Inhibition of TORC1 has no detectable effect on overall PKA activity. Wild type (PKA) and pka-as cells were grown to logarithmic phase in YPD medium (2% glucose) and then either DMSO or rapamycin (200 nM) or 1-NM-PP1 (25 μM) was added to the cultures. a Whole cell extracts were prepared from aliquots of cells taken after 0, 1, 2 and 3 h and were analyzed by Western blotting using an anti-PKA substrate antibody and actin antibody (loading control). b RNA was extracted from aliquots of DMSO and rapamycin-treated cells taken after 0, 1, 2 and 3 h and the expression of DIP5 and GAP1 was analyzed by Real-Time qRT-PCR. Data are presented as means ± standard deviation (n = 2 technical replicates). Fig. S7. TORC1 regulates the expression of glucose-responsive genes independently of Sch9. Wild type and sch9Δ cells were grown to logarithmic phase in SC/EG medium and then glucose (2% final concentration) was added to the cultures in the presence of either rapamycin (200 nM) or DMSO. Aliquots of the cultures were taken after 0’, 30’, 60’ and 120’. RNA was extracted from the cultures and the expression of the indicated 7 TGC genes (GFD2, GPG1, UGA1, RME1, CIT1, CRC1 and DHR2) were analyzed by Real-Time qRT-PCR. Data from these two additional biological replicates 2 and 3 are presented as means ± standard deviation (n = 2 technical replicates). Fig. S8. Growth of sch9Δ strain is impaired. Wild type and sch9Δ cells were diluted to a starting OD=O.2 in SC/D (SC medium containing 2% glucose) and the growth of the cultures was monitored by measuring the OD600 nm of the cultures every 2 hours for 10 hours. Data are presented as means ± standard deviation (n = 2). Fig. S9. Regulation of glucose-responsive genes by TORC1 is dependent on Tap42. Wild type or tap42-11 cells were grown to logarithmic phase at 25 °C (permissive temperature) in SC/EG medium and then shifted to 37 °C for 30’ to inactivate tap42-11. Glucose (2% final concentration) was added to the cultures in the presence of either rapamycin (200 nM) or DMSO. Aliquots of the cultures were taken after 0’, 30’, 60’ and 120’. RNA was extracted from the cultures and the expression of the 7 TGC genes (GFD2, GPG1, UGA1, RME1, CIT1, CRC1 and DHR2) were analyzed by Real-Time qRT-PCR. Data from these two additional biological replicates 2 and 3 are presented as means ± standard deviation (n = 2 technical replicates). Fig. S10. TORC1 regulates the glucose-responsive genes by inhibiting Sit4 and Rrd1/Rrd2 proteins. Wild type, sit4Δ, rrd1Δ, rrd2Δ, and rrd1Δ rrd2Δ cells were grown to logarithmic phase and then treated with either rapamycin (200 nM) or DMSO. Aliquots of the cultures were taken after 0’, 30’ and 60’. RNA was extracted from the cultures and the expression of the 7 TGC genes (GFD2, GPG1, UGA1, RME1, CIT1, CRC1, DHR2 and GPG1) were analyzed by Real-Time qRT-PCR. D1/R1 and D2/R2 represent samples from DMSO-treated/ Rapamycin-treated cells after 30’