Figure 5.
ADAR1 promotes lenalidomide resistance in LenR cells. (A) Immunofluorescence analysis of cellular dsRNA with J2 antibody in isogenic KMS-11 and MM1.S LenR cells (top). Orange-red fluorescence indicates dsRNA signals, and blue fluorescence indicates DAPI staining. Scale bars represent 20 μm. Bar graph quantification of the relative mean cytoplasmic dsRNA fluorescence intensity (bottom). (B) Sanger sequencing chromatograms illustrating the RNA editing levels of an ADAR1 target gene (SRP9) in isogenic KMS-11 and MM1.S LenR cells. Percentages denote the editing frequencies of the corresponding editing site indicated by the arrow. (C) Relative mRNA expression of CXCL10, IFN-α, and IFN-β in isogenic KMS-11 and MM1.S LenR cells after treatment with lenalidomide (10 μM) for 72 hours, determined by qRT-PCR. (D) Western blot validation of the effect of ADAR1 shRNA KD in KMS-11 and MM1.S LenR cells. Blot with ADAR1 p150 and ADAR1 p110 was probed with ab88574 (Abcam), and the blot with only ADAR1 p150 was immunoblotted with ab126745 (Abcam). (E) Cell viability of ADAR1-KD KMS-11 and MM1.S LenR cells at 6 days after treatment with increasing concentration of lenalidomide, determined by CTG assay. (F) Western blot analysis of whole-cell lysates for total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), and the dsRNA sensors-mediated signaling in ADAR1-KD KMS-11 and MM1.S LenR cells after treatment with lenalidomide (1 μM 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. (G) Heat map showing the normalized mRNA expression of ISGs and (IFNs: IFN-α and IFN-β) in ADAR1-KD KMS-11 and MM1.S LenR cells treated with lenalidomide (10 μM) for 72 hours, determined by qRT-PCR. Data are presented as mean ± SD of biological triplicates. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01, as determined by two-tailed Student t test.

ADAR1 promotes lenalidomide resistance in LenR cells. (A) Immunofluorescence analysis of cellular dsRNA with J2 antibody in isogenic KMS-11 and MM1.S LenR cells (top). Orange-red fluorescence indicates dsRNA signals, and blue fluorescence indicates DAPI staining. Scale bars represent 20 μm. Bar graph quantification of the relative mean cytoplasmic dsRNA fluorescence intensity (bottom). (B) Sanger sequencing chromatograms illustrating the RNA editing levels of an ADAR1 target gene (SRP9) in isogenic KMS-11 and MM1.S LenR cells. Percentages denote the editing frequencies of the corresponding editing site indicated by the arrow. (C) Relative mRNA expression of CXCL10, IFN-α, and IFN-β in isogenic KMS-11 and MM1.S LenR cells after treatment with lenalidomide (10 μM) for 72 hours, determined by qRT-PCR. (D) Western blot validation of the effect of ADAR1 shRNA KD in KMS-11 and MM1.S LenR cells. Blot with ADAR1 p150 and ADAR1 p110 was probed with ab88574 (Abcam), and the blot with only ADAR1 p150 was immunoblotted with ab126745 (Abcam). (E) Cell viability of ADAR1-KD KMS-11 and MM1.S LenR cells at 6 days after treatment with increasing concentration of lenalidomide, determined by CTG assay. (F) Western blot analysis of whole-cell lysates for total ADAR1 (ab88574; Abcam), ADAR1 p150 (ab126745; Abcam), and the dsRNA sensors-mediated signaling in ADAR1-KD KMS-11 and MM1.S LenR cells after treatment with lenalidomide (1 μM 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. (G) Heat map showing the normalized mRNA expression of ISGs and (IFNs: IFN-α and IFN-β) in ADAR1-KD KMS-11 and MM1.S LenR cells treated with lenalidomide (10 μM) for 72 hours, determined by qRT-PCR. Data are presented as mean ± SD of biological triplicates. Statistical significance is denoted by ∗P ≤ 0.05; ∗∗P ≤ 0.01, as determined by two-tailed Student t test.

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