Fig. 5: XL44 induces apoptosis in an hRpn13-dependent manner and reduces hRpn13Pru levels.
From: A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins
a–c Lysates from RPMI 8226 WT a–c, trRpn13-MM2 a, b or trRpn13-MM2 with reintroduced hRpn13 (b, trRpn13-MM2/hRpn13) treated for 24 h with 20 μM (a and b) or indicated concentration c of XL44 or DMSO (control) were immunoprobed as indicated. *, cleaved caspase-9. Cartoon (a, bottom) displaying the hRpn13 domains (purple circles), including hRpn2-binding Pru and UCHL5-binding DEUBAD, with the intrinsically disordered region as a black line. A red ‘X’ denotes the deletion of DEUBAD from hRpn13Pru. d Immunoblots as indicated of lysates from RPMI 8226 cells treated for indicated times with 20 μM XL44 or DMSO (0 h, vehicle control). Abundance (mean ± SD) plotted against XL44 concentration (c, μM) or time (d, hours) derived as a percentage by intensity ratio of hRpn13Pru normalized to β-actin (IhRpn13Pru/Iβ-actin)sample divided by DMSO-treated values and multiplied by 100 for two replicates. Data fit by ‘[Inhibitor] vs. normalized response - Variable slope’ (c) or ‘One phase decay’ (d) in GraphPad Prism9. DepC50 (c) and d1/2 (d) values are included, as are values for XL5-VHL-221. e MTT assay for RPMI 8226 WT (blue) or trRpn13-MM2 (light blue) cells treated for 48 h with indicated XL44 concentration. Data represent mean ± SD of n = 6 biological replicates. Viability calculated as (λ570)sample/(λ570)control*100 (%). f As in e, but with MCF7 (green), OVCAR-4 (purple) or OVCAR-5 (orange) cells. Data represent mean ± SD of n = 3 biological replicates. g MTS assay of human fibroblast 1634 (grey) or HS5 (BMSCs) (black) cells treated for 48 h with indicated XL44 concentrations. Data represent mean ± SD of n = 4 biological replicates. IC50 values in e, f analyzed by ‘[Inhibitor] vs. normalized response - Variable slope’ in GraphPad Prism9. h, LDH activity measured by CQUANT LDH Cytotoxicity Assay Kit for RPMI 8226 WT (blue) or trRpn13-MM2 (light blue) cells treated for 24 h with indicated XL44 concentration. Data represent mean ± SD of n = 4 biological replicates. Cytotoxicity calculated as ((LDH activity)sample-(LDH activity)spontaneous))/((LDH activity)Maximum-(LDH activity)spontaneous))*100 (%). Source data are provided as a Source Data file.
