Fig. 7: XL44 dually targets hRpn13 and PCLAF.

a Immunoblots of hRpn13 from 640 nM hRpn13 incubated without or with 30 nM commercial 26 S proteasome at 37 °C for 10 min. b, e Immunoblots of hRpn13 b, PCLAF b, RRM2 e, and β-actin b, e from lysates of RPMI 8226 cells pre-treated for 2 h with 100 nM proteasome inhibitor carfilzomib (CZ), 1 μM UAE inhibitor MLN7243 (UAEi) or 1 μM NAE inhibitor MLN4924 (NAEi), followed by 22 h treatment with 20 μM XL44 or DMSO. c Schematic representation of hRpn13 proteolysis (displayed as scissors) by proteasomes to generate hRpn13^Pru and its clearance by XL44. XL44-induced depletion of hRpn13^Pru requires UAE and proteasome activity, with hRpn13^Pru proteolysis followed by ubiquitin-dependent degradation. hRpn13 domains and intrinsically disordered regions are represented by purple circles and black lines, respectively. d Schematic representation of degradation of PCLAF and RRM2 by proteasomes, as accelerated by XL44. Intrinsically disordered region (bold line), domain (black oval), and KEN box (yellow) are displayed, respectively. f Lysates immunoprobed for PCLAF and β-actin from HCT116 cells transfected with 1 μg empty vector (control), HA-PCLAF or HA-PCLAF (AA, with K78A and E79A substitutions), followed 48 h later by treatment with 20 μM XL44 or DMSO for 24 h. g Cell metabolism measured by an MTT assay for HCT116 WT or trRpn13 cells transfected with 50 nM scramble or PCLAF siRNA for 48 h, followed by 24 h treatment with 20 μM XL44 or DMSO (vehicle control). Data represent mean ± SD of n = 6 biological replicates. ****P < 0.0001; **P < 0.004; P (WT Scramble vs. WT PCLAF) = 0.0018; P (WT Scramble vs. trRpn13 Scramble) = 0.0029; P (trRpn13 Scramble vs. trRpn13 PCLAF) = 0.0031; P (WT Scramble vs. trRpn13 PCLAF) < 0.0001; ‘one-way ANOVA’ (Tukey’s multiple comparisons test) in GraphPad Prism9. Viability is calculated as (λ₅₇₀)_sample/(λ₅₇₀)_control*100 (%). The experiments in 7a, 7b, 7e and 7 f were performed twice. Source data are provided as a Source Data file.