(A) Uninfected BJABs, or cells infected with a lentiviral construct containing non-targeted control (NTC) or UBQLN1- shRNA (construct #1 from Figure 1—figure supplement 1) were incubated with 100 ng/mL doxycycline (+dox) or vehicle for 72 hr, following which UBQLN1 and Tubulin were detected by western blot. (B) BJAB cells stably transfected with UBQLN1- (U, open symbols) or an NTC-shRNA (N, filled symbols) were incubated with doxycycline (triangles) or vehicle (squares) for 4 days. Viable cells were measured with the Cell Titer Glo (Promega) assay in duplicate with mean ±SEM, and luminescence quantified as arbitrary units. One representative experiment of two is shown. (C) Untransfected HeLa cells, or cells stably transfected with NTC- or UBQLN1 shRNA were treated similarly to BJABs and western blotted as in Figure 1A. (D) Viable cells were measured as in (Figure 1B) for HeLa cells. (E) BJAB cells containing either an NTC- or UBQLN1 shRNA construct (#1) were cultured in the presence of 100 ng/mL doxycycline starting at day 2 and then washed to remove doxycycline (grey-shaded box). The number of viable cells was estimated by CellTiter Glo as arbitrary units of luminescence in duplicate wells. Each line represents the average of duplicate wells with mean ± SEM. (F) Isolated cytosolic protein from NTC- or UBQLN1- shRNA expressing BJABs was labeled in triplicate with TMT and analyzed by mass spectrometry. Shown are the roughly 1300 significantly altered (p<0.05) proteins and their relative enrichment in UBQLN1-depleted cytosol. Mitochondrial proteins, as listed in Mitocarta 2.0 (Calvo et al., 2016), are shown in white. Grey box highlights proteins that were at least twofold accumulated in KD cytosol (92 proteins), which were used for DAVID pathway analysis. (G) Proteins identified in (F) were plotted according to their GRAVY score against their relative enrichment in UBQLN1-depleted cytosol. As in (F), mitochondrial proteins are shown in white. (H) Cellular fractionation of BJAB cells after 48 hr with 100 ng/mL doxycycline. Cells were fractionated according to Materials and Methods. Samples were blotted for mitochondrial proteins VDAC and ATP5G1, as well as for Tubulin. ATP5G1 ran at 17 kDa and thus represents the precursor form of the protein before mitochondrial insertion. N: Non-targeting shRNA construct. U: UBQLN1 shRNA construct. Shown is one representative experiment of three. (I) Quantification of ATP5G1 in cytosolic fractions of BJABs from three independent experiments. ATP5G1 was normalized to Tubulin in each sample. Data shown are mean ±SEM, and significance was determined with an unpaired Student’s T test.