Fig 2: Modulation of Pol III subunit levels as a consequence of Pol III inhibition by MPA. (A) Wild-type yeast cells expressing HA-tagged C160 subunit were grown in minimal medium without uracil to exponential growth phase at 30°C and then treated with 100 μg/ml MPA for 2 h. (Top) Representative Western blot showing the levels of indicated Pol III subunits and Pgk1, which was used as a loading control. (Bottom) Quantification of Pol III subunit levels. The mean subunit expression is shown normalized to the Pgk1 protein, and the time zero sample is set to 1. n = 3. Error bars represent the standard deviation. (B) Plot comparing the kinetics of tRNA decrease and C160 level downregulation upon MPA treatment. The data for selected tRNA levels from Fig. 5 were used. The data related to C160 levels were taken from the work of Leśniewska et al. (27) and were obtained from the same strain, MW4415, grown and treated similarly as in panel A. The error bars were omitted for the sake of figure clarity. (C) Wild-type yeast cells expressing HA-tagged C160 and GFP-tagged AC40 subunits were grown in minimal medium without uracil to exponential growth phase at 30°C and then left untreated (NT) or treated with either ethyl alcohol (EtOH) or 100 μg/ml MPA for 2 h. Cellular extracts were incubated with magnetic beads coated with anti-GFP antibody. (Top) After extensive washes, immunoprecipitated proteins were eluted and analyzed by Western blotting using antibodies to GFP, HA tag, C82, and C53. (Bottom) Estimation of binding of C160, C82, and C53 to AC40. The binding of the subunits in MPA-treated cells was calculated relative to their binding in EtOH-treated cells, which was set to 1. n = 3. Error bars represent the standard deviation.