Figure 4.
ADAR1-mediated RNA editing inhibits lenalidomide-induced dsRNA-sensing responses in MM cells. (A) Immunofluorescence analysis of cellular dsRNA with J2 antibody in Len-sensitive (KMS-11 and MM1.S cells) vs Len-resistant (RPMI-8226 cells and OCI-MY5) cells after treatment with lenalidomide (1 or 10 μM) for 72 hours (left). Orange-red fluorescence indicates dsRNA signals and blue fluorescence indicates DAPI (4′,6-diamidino-2-phenylindole) staining. Scale bars indicate 20 μm. Bar graph quantification of the relative mean cytoplasmic dsRNA fluorescence intensity (right). (B) Dot blot analysis of cellular dsRNA using dsRNA-specific J2 antibody in total RNA extracted from ADAR1-KD RPMI-8226 and OCI-MY5 cells blotted on Biodyne nylon membrane. RNA loading was verified by methylene blue staining. (C) Dot blot analysis of cellular dsRNA using dsRNA-specific J2 antibody in total RNA extracted from ADAR1-KD RPMI-8226 and OCI-MY5 cells blotted on Biodyne nylon membrane. As indicated, total RNA extracted were treated with RNase III for 30 minutes at 37°C as negative control for dsRNA signal. RNA loading was verified by methylene blue staining. (D) Immunofluorescence analysis of cellular dsRNA with J2 antibody in ADAR1-KD RPMI-8226 cells after treatment with lenalidomide (10 μM) for 72 hours (left). Orange-red fluorescence indicates dsRNA signals, green fluorescence indicates ADAR1 p150 expression, and blue fluorescence indicates DAPI staining. Scale bars indicate 20 μm. Bar graph quantification of the relative mean cytoplasmic dsRNA fluorescence intensity (right). (E) Western blot analysis of whole-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 after treatment with lenalidomide (10 μM) for 72 hours (top). The bar graphs (bottom) 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) Induction of IFN-β protein secretion by ADAR1-KD RPMI-8226 and OCI-MY5 cells after treatment with lenalidomide (1 or 10 μM) for 72 hours, assessed by ELISA. 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.

ADAR1-mediated RNA editing inhibits lenalidomide-induced dsRNA-sensing responses in MM cells. (A) Immunofluorescence analysis of cellular dsRNA with J2 antibody in Len-sensitive (KMS-11 and MM1.S cells) vs Len-resistant (RPMI-8226 cells and OCI-MY5) cells after treatment with lenalidomide (1 or 10 μM) for 72 hours (left). Orange-red fluorescence indicates dsRNA signals and blue fluorescence indicates DAPI (4′,6-diamidino-2-phenylindole) staining. Scale bars indicate 20 μm. Bar graph quantification of the relative mean cytoplasmic dsRNA fluorescence intensity (right). (B) Dot blot analysis of cellular dsRNA using dsRNA-specific J2 antibody in total RNA extracted from ADAR1-KD RPMI-8226 and OCI-MY5 cells blotted on Biodyne nylon membrane. RNA loading was verified by methylene blue staining. (C) Dot blot analysis of cellular dsRNA using dsRNA-specific J2 antibody in total RNA extracted from ADAR1-KD RPMI-8226 and OCI-MY5 cells blotted on Biodyne nylon membrane. As indicated, total RNA extracted were treated with RNase III for 30 minutes at 37°C as negative control for dsRNA signal. RNA loading was verified by methylene blue staining. (D) Immunofluorescence analysis of cellular dsRNA with J2 antibody in ADAR1-KD RPMI-8226 cells after treatment with lenalidomide (10 μM) for 72 hours (left). Orange-red fluorescence indicates dsRNA signals, green fluorescence indicates ADAR1 p150 expression, and blue fluorescence indicates DAPI staining. Scale bars indicate 20 μm. Bar graph quantification of the relative mean cytoplasmic dsRNA fluorescence intensity (right). (E) Western blot analysis of whole-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 after treatment with lenalidomide (10 μM) for 72 hours (top). The bar graphs (bottom) 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) Induction of IFN-β protein secretion by ADAR1-KD RPMI-8226 and OCI-MY5 cells after treatment with lenalidomide (1 or 10 μM) for 72 hours, assessed by ELISA. 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.

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