Fig. 4: At 24 °C, TLC1 levels are higher in pop cells, while Est1 and Est2 levels are similar to WT.

Protein extracts from the indicated strains grown at 24° or 30 °C were analyzed by western blotting using a MYC-antibody to detect a Est1-MYC or c Est2-MYC. [(Est3 levels were not determined because we do not have an epitope tagged allele of Est3 that has WT telomere length and that can be detected by westerns in whole cell extracts⁴]. Levels of b Est1-MYC and d Est2-MYC from three biological replicates (black circles) grown at 24 °C were quantified after being normalized to levels of tubulin in the same samples. The normalized value of each protein in WT cells was defined as one. At 30 °C, levels of Est1-MYC and Est2-MYC in pop cells were normalized only to WT levels of Est1 or Est2 as tubulin and Pgk1 levels were also lower at 30 °C. The fold difference in Est1-MYC or Est2-MYC in pop1 and pop6 cells compared to WT is shown. The loading control (LC) for the Est1-MYC western at 30 °C was Pgk1. For all other westerns, the LC was tubulin. e Total RNA was extracted from cells grown at 24 °C or 30 °C for ~50 generations. RNA levels were determined by RT-qPCR for e TLC1 or f NME1 in WT (gray bars), pop1 (blue bars) and pop6 (purple bars). Individual biological replicates are shown (black circles). TLC1 and NME1 levels were normalized to levels of ACT1 mRNA in the same samples using the 2^-ΔΔCt method⁵⁶. At both 24 and 30 °C, the increase in TLC1 (and the decrease in NME1) RNA in pop cells was suppressed by a WT copy of the mutated POP gene. Error bars are one standard deviation from the average value of three or more independent experiments. P-values were calculated using unpaired two-tailed Student’s t-test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; NS, not significant, P >0.05. Source data are provided as a Source Data file.