acted with RNeasy-Kit (Qiagen). 500 ng of RNA were reverse-transcribed to cDNA by the use of reverse transcriptase and oligo(dT) primer in line with standard protocols. For the following PCR reaction, 1 fiftieth of the obtained cDNA was applied in combination together with the following oligos: RUVBL1-fwd (5′-CTG TGT CAT CAG AGG CAC TGA-3′), RUVBL1-rev (5′-AAG TTC ACT GAT CTC TTC GAC ATG-3′); RUVBL2-fwd (5′-CAT CAC GCG AAT CCG G-3′), RUVBL2-rev (5′-TGA GTA GAC CCG CTT GAT GTC-3′); GAPDH-fwd (5′-CTC CTC TGA CTT CAA CAG CGA CAC-3′), GAPDH-rev (5′-CTC TCT CTT CCT CTT GTG CTC TTG C-3′). Clonogenic survival assay was performed as previously described [57]. Media have been replaced each 4 days to ensure a continual doxycycline concentration (1 g/ml). The assays were performed in triplicates. Propidium Iodide staining and flow cytometric evaluation were performed as previously described [58].
S1 Fig. Specificity of RUVBL1 staining. (A) Specificity from the RUVBL1 staining was ascertained by pre-incubating the antibody with 2224-02-4 chemical information recombinant His-RUVBL1 for 1h (His-RUVBL1: antibody, 10:1). Phase contrast pictures had been taken as handle. A merged image is shown with RUVBL1 (green) and DAPI (blue). (B) U2OS cells were transfected with RUVBL1 distinct siRNA oligos 48 h prior fixation and staining with anti-RUVBL1 antibody. DNA is counterstained with DAPI (blue). (C) A pattern equivalent to that observed inside a was obtained working with a different anti-RUVBL1 antibody. (PDF) S2 Fig. RUVBL1 depletion provides rise to lagging chromosomes. U2OS T-REx cells stablytransfected using a doxycycline-inducible shRNA against endogenous RUVBL1 had been co-transfected using a doxycycline-inducible shRNA-resistant FLAG-tagged murine RuvBL1 construct and 10205015 treated or not with doxycycline for 48 h, as indicated. Protein expression was verified by immunoblotting (A) and occurrence of lagging chromosomes was quantified by analyzing 75 anaphases for each cell line and condition (B). (PDF) S3 Fig. Sequence alignment of RUVB-like proteins. (A) Protein sequences from human RUVBL1 (NP_003698) and RUVBL2 (NP_006657) have been obtained from http://www.ncbi.nlm. nih.gov and aligned with http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi working with default parameters. Alignment was processed making use of Boxshade three.two, with identical amino acids in black and homologous amino acids in gray boxes. The sequence was colored as outlined by the domain structure, with domain 1 in orange, domain two in blue and domain 3 in red, respectively. Walker A and Walker B motifs are highlighted with black rectangles and possible PLK1 phosphorylation motifs with red rectangles, respectively. (B) Sequence comparison of human RUVBL1 with RuvB of Thermotoga maritima (AAB03727). (C) The structure of RUVBL1 is shown with domains highlighted within the colors utilised above. Threonine at position 239 in RUVBL1 is highlighted in turquoise. The structure was modified depending on published data [10] utilizing PyMOL software program plus the PBD files 2c9o (for RUVBL1) and 1in7 (for RuvB), respectively. (PDF) S4 Fig. In vitro phosphorylation of RUVBL1 by PLK1. (A) Various amounts of purified His-tagged RUVBL1 were incubated with PLK1 within the presence of [-32P]ATP. Casein served as optimistic manage. Proteins had been separated by SDS-PAGE plus the Coomassie blue-stained gel was subjected to autoradiography. (B) His-tagged RUVBL1 mutants were purified to near homogeneity and subjected to SDS-PAGE and Coomassie blue staining. (C) RUVBL1 is often phosphorylated although in complex with RUVBL2. GST-tagged
