The impact of CRBN mutations on neosubstrate degradation after IMiD/CELMoD treatment. Approximately 1 × 10⁶ cells from each MM1.s^CRBNKO clone 1 cell line (expressing a differently mutated CRBN, CRBN^WT, or EV) and the parental MM1.s cell line were drug treated (10 μM Len, 1 μM Pom, 0.1 μM Iber, 0.01 μM Mezi, or DMSO) for 24 hours before harvesting for either western blotting or RNA extraction. (A) Immunoblotting results for the neosubstrate protein Aiolos in the investigated cell lines after IMiD/CELMoD treatment. For each cell line, protein-level measurements for all treatments are calculated as fold change normalized to the corresponding DMSO-treated control. The color of the title for each blot denotes the effect of corresponding mutation on protein levels, where green is associated with expression comparable with parental MM1.s (shown in lower right corner), red denotes no reduction in protein levels, and orange highlights the mutations whose presence leads to partial reduction in protein levels. Blots shown are representative of 3 biological replicates. (B) Optical densitometry quantification of the immunoblotting results shown in Figure 3A for the 3 biological replicates. Data are shown as mean ± SEM. (C) Quantitative reverse transcription polymerase chain reaction results for the messenger RNA expression levels of transcription factor IRF4 after IMiD/CELMoD treatment. Data are shown as mean ± SEM of n = 3 biological repeats.