Figure 3. Transcriptional repression of Inh1 maintains ∆ψ_m, ISC synthesis, and proliferation of ρ⁰ cells.

1. qRT–PCR analysis of INH1 mRNA levels. Data are mean ± SD from three biological replicates.
2. Western blot analysis of Inh1 protein levels. Whole cell lysates were extracted, and equal amount of proteins was loaded for Western blot.
3. Deletion of INH1 benefits the proliferation of ρ⁰ cells at 37°C. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30°C and 37°C for 2 days.
4. Western blot analysis of copper‐inducible overexpression of Inh1. A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus. Inh1 overexpression was induced by 100 μM CuSO₄ for 12 h.
5. Overexpression of Inh1 decreases ∆ψ_m in ρ⁰ cells. Cells were cultured in SCD or SCD plus 100 μM CuSO₄ for 12 h to mid‐log phase. Cells were then stained with 125 nM TMRM for FACS analysis.
6. Overexpression of Inh1 represses the proliferation of ρ⁰ cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO₄ plates at 30°C for 2 days.
7. Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins (Nfs1 and Yah1) and nuclear ISC‐containing protein Pol3. Cells were cultured in SCD or SCD plus 100 μM CuSO₄ for 12 h to mid‐log phase. Whole cell lysates were extracted, and equal amount of proteins was loaded for Western blot. Nfs1 and Yah1 were endogenously tagged with FLAG, and Pol3 was endogenously tagged with HA.
8. Cartoon illustration of the translational repression of Hap4 and the subsequent transcriptional downregulation of Inh1 to maintain ∆ψ_m in ρ⁰ cells.

Data information: The red highlight two cell lines for comparison (E, F).

Source data are available online for this figure.