Figure 1.
Figure 1. MM cells with lower antioxidative capacity are vulnerable to lenalidomide-mediated cytotoxicity. (A) RPMI-8226, JJN3, MM.1S, and KMS11 cell lysates were prepared, separated by electrophoresis, and immunoblotted with the indicated antibodies: IKZF1, IKZF3, CRBN, and β-actin. β-Actin was used as a loading control. (B) MTT assay of MM cell lines showed that MM.1S was lenalidomide hypersensitive, KMS11 was sensitive, and RPMI-8226 and JJN3 were lenalidomide resistant. Each experimental condition was performed in triplicate and repeated at least twice. (C) Bubble assay depicts release of oxygen from H2O2 by MM cells, with higher antioxidative capacity being associated with lenalidomide resistance. An equal number (1 × 106) of cells in PBS from various cell lines was incubated with H2O2, and oxygen release was qualitatively measured. (D) H2O2-mediated intracellular oxidation of FADH2 and NAD(P)H quantitatively determined antioxidative capacity in MM cells. Flow cytometry of RPMI-8226, JJN3, MM.1S, and KMS11 cells in PBS, with or without H2O2 treatment (100 μM) (gated for 10 000 events), showed significantly increased FAD autofluorescence (FITC-A) and significantly decreased NAD(P)H autofluorescence in RPMI-8226 and JJN3 with H2O2; these changes were associated with greater antioxidative capacity and resistance to lenalidomide compared with MM.1S and KMS11 cells, which have lower antioxidative capacity and greater sensitivity to lenalidomide. (E-F) Mean fluorescence intensity from 3 independent experiments for FAD and NAD(P)H autofluorescence, respectively. Data are shown as mean ± standard error of the mean (SEM). (G) Antioxidative capacity and prediction of lenalidomide sensitivity for samples from patients with primary MM. CD138+ patient samples were treated with or without H2O2 (100 µM or control) for 30 minutes. Fluorescence readings were obtained for FAD at 450/535- and NAD(P)H at 350/460-nm excitation and emission wavelengths, respectively. Data are shown as mean ± SEM of 4 biological repeats. **P < .01 compared with control; ***P < .001 compared with control.

MM cells with lower antioxidative capacity are vulnerable to lenalidomide-mediated cytotoxicity. (A) RPMI-8226, JJN3, MM.1S, and KMS11 cell lysates were prepared, separated by electrophoresis, and immunoblotted with the indicated antibodies: IKZF1, IKZF3, CRBN, and β-actin. β-Actin was used as a loading control. (B) MTT assay of MM cell lines showed that MM.1S was lenalidomide hypersensitive, KMS11 was sensitive, and RPMI-8226 and JJN3 were lenalidomide resistant. Each experimental condition was performed in triplicate and repeated at least twice. (C) Bubble assay depicts release of oxygen from H2O2 by MM cells, with higher antioxidative capacity being associated with lenalidomide resistance. An equal number (1 × 106) of cells in PBS from various cell lines was incubated with H2O2, and oxygen release was qualitatively measured. (D) H2O2-mediated intracellular oxidation of FADH2 and NAD(P)H quantitatively determined antioxidative capacity in MM cells. Flow cytometry of RPMI-8226, JJN3, MM.1S, and KMS11 cells in PBS, with or without H2O2 treatment (100 μM) (gated for 10 000 events), showed significantly increased FAD autofluorescence (FITC-A) and significantly decreased NAD(P)H autofluorescence in RPMI-8226 and JJN3 with H2O2; these changes were associated with greater antioxidative capacity and resistance to lenalidomide compared with MM.1S and KMS11 cells, which have lower antioxidative capacity and greater sensitivity to lenalidomide. (E-F) Mean fluorescence intensity from 3 independent experiments for FAD and NAD(P)H autofluorescence, respectively. Data are shown as mean ± standard error of the mean (SEM). (G) Antioxidative capacity and prediction of lenalidomide sensitivity for samples from patients with primary MM. CD138+ patient samples were treated with or without H2O2 (100 µM or control) for 30 minutes. Fluorescence readings were obtained for FAD at 450/535- and NAD(P)H at 350/460-nm excitation and emission wavelengths, respectively. Data are shown as mean ± SEM of 4 biological repeats. **P < .01 compared with control; ***P < .001 compared with control.

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