Fig 2: iP activity makes a significant contribution to UPS capacity and undergoes selective and coordinated recovery from inhibition.(A) Ubiquitin western blot and matched activity assay for the SUP-B15 cell line (with approximately 80:20 LMP7: β5 activity) showing a substantial increase in global ubiquitination levels upon one-hour pulse treatment with ONX 0914. A significant increase in ubiquitination was not observed with PR-825 treatment, suggesting that LMP7 is a more dominant contributor to UPS capacity in this cell line in accordance with the proportionally higher LMP7 activity level. Selective recovery of LMP7 activity over 24 hr in the (B) SUP-B15 and MOLT-4 cell lines and in (C) primary PBMCs from a healthy donor (see also Figure 5—figure supplement 2) upon one-hour pulse treatment with ONX 0914. The SUP-B15 and MOLT-4 lower panels show the fold change in LMP7 and β5 activity levels normalized to the one-hour time point. For all activity assays, mean activity is reported with error bars representing the standard deviation from n = 3–6 replicates. (D) Corresponding qRT-PCR analysis of PSMB8 (LMP7) and PSMB5 (β5) mRNA dynamics in the cell lines and PBMCs following ONX 0914 treatment. Mean expression levels are reported with normalization to GAPDH. Statistically significant differences are indicated (*p≤0.05) as determined using a Student’s t-test with error bars representing the SEM from n = 3 replicates. (E) Corresponding western blots demonstrating accumulation of processed Nrf1 (p110) in RIPA lysates from the cell lines and PBMCs following ONX 0914 treatment. Full-length (FL) Nrf1 was observed in the PBMC lysates. .

Fig 3: In vitro analysis of proteasome subunit expression and sensitivity to proteasome inhibitors.(A) Proteasome subunit expression in breast cancer cell lines (Neve et al, 2006). (B) Inducible-to-constitutive subunit expression ratios (i:c) across molecular breast cancer subtypes (stats: two-way ANOVA). (C) Linear regression and Pearson correlation analysis of the relationships between i:c and sensitivity to bortezomib or carfilzomib (LD50, lethal dose 50%). Correlation coefficients (r) and regression fit values (r2) indicated. (D) Western analysis of inducible subunits and the PA28 cap in lines with a range of bortezomib sensitivities. #MCF10A is a spontaneously immortalized breast-derived line included for comparison. (E) Native in-gel proteasome activity assay with lysates from MDA-MB-468 and MCF7 with/without 2-h bortezomib treatment. (F) Light microscope images of MDA-MB-468 and MCF7 48 h after bortezomib treatment (captured at 20× magnification, scale bar 50 µm). (G) Cell viability after siRNA-mediated depletion of PSMB8 (i) or PSMB5 (ii). P-values in this figure were from unpaired, two-tailed t tests (pair-wise comparisons) or one-way ANOVA tests (comparison across multiple groups): *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Source data are available for this figure.