Fig. 3

RHM treatment counteracts LIG3 activity and induces DNA damage in MM cells. KMS26 and AMO1 cells were treated with increasing dose of RHM or vehicle. A: Immunoblot analysis of DDR markers was performed 48 h after treatment. B: γ-H2AX foci evaluation by immunofluorescence 48 h after treatment. Representative images of unrepaired DSBs are shown. DAPI (blue) was used for nuclear staining. C. Immunoblot analysis of nuclear soluble and chromatin bound fractions prepared from AMO1 cells treated with or without RHM (5 μM). D. KMS26 and AMO1 cells were treated with RHM (5 μM) or vehicle. Left: Mitochondrial DNA copy number, as measured by qRT-PCR 48 h after treatment. Right: Mitochondria were stained with Mitotracker Red and visualized by fluorescence microscopy 48 h after treatment. E. Alt-NHEJ repair was evaluated by EJ2- GFP assay on AMO1 cells 72 h after treatment with RHM (5 μM) or vehicle. F. Affymetrix CytoScan HD Array analysis, using genomic DNA from AMO1 treated with RHM (2,5 μM) or vehicle. Representative images of deletions or gains acquisition on chromosome 16 (16p11.2) and 1(1p36.3), respectively. Red lines represent deletions, while blue lines represent gains Results are representative of three independent experiments ± SD. *, P < 0.01.