Fig 2: Multi time point pSILAC mass spectrometry.(a) Outline of the multi time point pulse-SILAC mass spectrometry experimental design. For simplification, replicate and reverse experiments are not depicted in this figure. (b) Scatter plot depicting the change of H/L protein ratios over time. Protein ratios of the 10 and 16 h time points are compared to the 6 h time point. Data in this figure are presented as means of biological replicates for T6 and T16 (n=2) or individual data points for T10 (n=1). (c) Frequencies of H/L protein ratios at different time points (data as in b). (d) Plots depicting the logarithmic H/L protein ratios over time are shown for the two validated targets CSNK1A1 and ZFP91 as well as for control proteins GAPDH, UBA1, DDB1 and CopS5. Differential turnover for CSNK1A1 and ZFP91 is observed, while stable conditions are found for controls. Data in this figure are presented as individual data points and separate r2 values for the linear regression are provided for lenalidomide and DMSO samples. (e) Scatter plot comparing protein half-lives of lenalidomide treated and control samples. Protein half-lives were obtained by fitting the H/L protein ratios of T6 (n=2), T10 reverse (n=1) and T16 (n=2) time points to a decay function24. For further analysis, we retained only proteins quantified in all samples and with r2>0.9 for both linear regression fits (DMSO and lenalidomide treated), resulting in a total of 2,759 proteins. CSNK1A1 was found to exhibit a reduced half-life in presence of lenalidomide. Blue and red dotted lines indicate ±3 and ±5 s.d., respectively. ZFP91 was dropped from the data analysis for missing values in two replicate measurements. The depicted delta-half-life for ZFP91 is calculated using imputed values from replicates to replace the missing values. (f) Scatter plot depicting the negative correlation between differences in protein half-life (data as in e), and differences in log2 H/L protein ratios at the T16 time point (LENA-DMSO, mean of two biological replicates). R corresponds to the Pearson's correlation coefficient. (g) Fold change in H/L ratios comparing lenalidomide to DMSO control treatment on the x axis (HEK293 T16 SILAC samples). Moderated t-test P values were calculated using the limma package and shown as -log10 values on the y axis. The vertical dashed lines indicate ±5 s.d. log2 fold change in H/L ratio and the horizontal dashed line indicates P value <0.001. Data shown represents two biological replicates.

Fig 3: ZFP91 and IKZF1/3 share a common sequence motif.(a) In vitro ubiquitination of recombinant ZFP91 by recombinant N8CRL4ACRBN is facilitated by lenalidomide (lanes 5–7), thalidomide (lane 8) and pomalidomide (lane 9). Shown is one representative experiment out of two replicates. (b) Alexa488-N8CRL4ACRBN titrated to biotinylated wild-type ZFP91 at 100 nM in the presence of lenalidomide or DMSO as a control in presence of tracer Tb-streptavidin at 2 nM. Data are presented as means±s.d. (n=3). (c) Multiple sequence alignment of the putative ZFP91, IKZF1 and IKZF3 degron motifs11. Identical amino acids, and structural residues of the ZnF motif, are highlighted in black and grey, respectively. (d) Titration of thalidomide, lenalidomide and pomalidomide to Alexa488-N8CRL4ACRBN at 0.2 µM, biotin-ZFP91 at 0.1 µM and Tb-streptavidin at 2 nM. EC50 values are shown and indicate preference for pomalidomide in vitro. Data are presented as individual data points for one representative experiment out of four replicates. (e) MM.1S cells were treated with increasing concentrations of lenalidomide, thalidomide, pomalidomide or a DMSO control for 12 h. Co-treatment with the proteasome inhibitor bortezomib was included as an additional control. ZFP91 and GAPDH levels were detected using anti-ZFP91 or anti-GAPDH immunoblotting (shown is the data for one representative experiment).

Fig 4: Validation of ZFP91 as bona fide lenalidomide-induced CRL4CRBN substrate.(a) MM.1S cells were treated with increasing concentrations of lenalidomide or with DMSO. Following 24 h of incubation, ZFP91 and GAPDH levels were detected by anti-ZFP91 and anti-GAPDH western blot (shown is one representative experiment out of three replicates). (b) HEK293T cells were treated with 50µg ml-1 cycloheximide and increasing concentrations of lenalidomide, thalidomide or with DMSO, and cells were incubated for 6 h. ZFP91 and GAPDH levels were detected using anti-ZFP91 or anti-GAPDH immunoblotting (shown is one representative experiment out of five replicates). (c) as in (b) but using SK-N-DZ cells instead (shown is one representative experiment out of three replicates). (d) as in (b) but with co-treatment of bortezomib (proteasome inhibitor, lanes 4, 5) or MLN4924 (inhibitor of the NEDD8-activating enzyme, lanes 6,7). Shown is one representative experiment out of three replicates. (e) as in (b) using parental HEK293T (lane 1–3) or two independent pools of HEK293T cells with genetic inactivation of CRBN by CRISPR/Cas9 (shown is one representative experiment out of two replicates).