Lenalidomide induces dsRNA-sensing pathway and stimulates anti-MM intrinsic immunity in MM cells. (A) Schematic diagram illustrating the activation of the dsRNA sensor–mediated signaling pathways in cells. (B) Western blot analysis of cytoplasmic cell lysates for total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), and the dsRNA sensor–mediated signaling in ADAR1-KD RPMI-8226 and OCI-MY5 cells (left). The bar graphs (right) summarize p-PKR, p-IRF3, and p-eIF2α band intensities normalized to GAPDH and total protein content, quantified via ImageJ. Densitometry analyses for the relative protein expression normalized to GAPDH are displayed below the western blots. (C) Spontaneous IFN-β protein secretion by ADAR1-KD RPMI-8226 and OCI-MY5 cells in the culture supernatant, assessed by enzyme-linked immunosorbent assay (ELISA). (D) Heat map showing the normalized mRNA expression of ISGs and IFNs (IFN-α and IFN-β) in ADAR1-KD RPMI-8226 and OCI-MY5 cells, determined by qRT-PCR. (E) Western blot analysis of cytoplasmic-cell lysates for total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), and the dsRNA sensor–mediated signaling in lenalidomide-sensitive (Len-sensitive) KMS-11 and MM1.S cells vs LenR RPMI-8226 and OCI-MY5 cells after treatment with lenalidomide (1 or 10 μM) for 72 hours (left). The bar graphs (right) summarize p-PKR, p-IRF3, and p-eIF2α band intensities normalized to GAPDH and total protein content, quantified via ImageJ. Densitometry analyses for the relative protein expression normalized to GAPDH are displayed below the western blots. (F) Histogram of GO enrichment analysis after lenalidomide treatment (10 μM) in MM1.S and OPM2 cells at 12 hours and 24 hours, respectively, relative to control (RNA-seq data GSE246435, FDR adjusted P value <0.05 and log2-fold >1.5). Data are presented as mean ± SD of biological triplicates. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001, as determined by two-tailed Student t test. FDR, false discover rate; GO, gene ontology.