bio rad gene pulser xcell electroporation system  (Bio-Rad)

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: a, b Surface plasmon resonance (SPR) experiment showing direct binding of a C-terminal Ku80 fragment (aa 384 – 732) to surface-coupled EndoG. a Sensorgram of a representative single-cycle measurement with EndoG as surface-coupled ligand and the C-terminal Ku80 fragment as analyte, applied in increasing concentrations (10, 20, 40, 80, 160, and 320 nM) (red line). The maximal response at equilibrium was marked for each concentration (black x). b Response-concentration plot from (a) shows the equilibrium response of each analyte concentration (black circles). A one-site binding fit (red line) was used to determine the dissociation constant K D . The measurement was done in triplicate, resulting in a mean K D and standard deviation of 72.7 ± 13.2 nM. c Structure of Ku80 variants analyzed. Ku80-wt comprises the N-terminal Von Willebrand A domain , the central DNA end-binding core (aa 210–531) , which further encompasses aa 449–477 heterodimerizing with Ku70 and is followed by a nuclear localization signal (NLS) . The structurally defined C-terminal domain (CTD, red) is predicted to form α-helices at aa 594–705 and an unstructured tail, which binds DNA-PKcs at the PIKK (PI3 kinase-like kinase) interaction motif (aa 718-732) , . For expression of Ku80-Ct, devoid of DNA, Ku70 and DNA-PKcs binding sites, a truncated CTD (aa 592-709) was fused to the NLS (aa 562–568). d Principle of MLL bcr rearrangement assay. Recombination measurements rely on quantification of EGFP-positivities post doxorubicin-treatment of K562 cells with chromosomally integrated reporter construct for recombination between differently mutated EGFP genes encompassing the 0.4 kbp therapy-related MLL bcr , . e Ku80 variant expression in K562. Proteins were extracted 24 h post-electroporation of K562 cells with expression constructs for Ku80 variants specified in (a) or empty vector (ctrl). Western blot analysis was performed using antibodies for total Ku80 and the N-terminal DDK/Flag-tag of ectopically expressed variants (representative from 2 independent experiments). Uncropped Western blots in Source Data (uncropped images). f , g Recombination measurements post-chemotherapeutic treatment. K562 reporter cells, ectopically expressing Ku80 variants for 24 h, were treated with 2.0 μM doxorubicin (blue) for 4 h followed by 72 h drug-free culture, with solvent (DMSO, white) or 10 µM etoposide (gray) for 4 h followed by 72 h drug-free culture or for 72 h with etoposide and FACS analysis. Recombination (rec.) frequencies of EGFP-positive living cells in the population were normalized to the mean of ctrl values set to 100% per experiment (average for doxorubicin: 2 × 10 −5 ; for etoposide, 4 h: 6 × 10 −5 and 72 h: 5 × 10 −4 ) to calculate relative rec. frequencies. Data are presented as mean + SEM. Significances were calculated by the Kruskal-Wallis H-test followed by a two-tailed Mann-Whitney-U test and values of p < 0.05 indicated, gating strategies in Supplementary Fig. , d. f Recombination measurements post doxorubicin treatment ( n = 6 samples from 3 independent experiments). g Recombination measurements post etoposide treatment. DMSO and etoposide (4 h): n = 6 samples from 3 independent experiments. Etoposide (72 h): n = 15 samples from 5 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Then, a total amount of 50 μg plasmid for expression of one of the Ku80 variants was added, and the mixture transferred into a Gene Pulser ® /Micropulser TM electroporation cuvette (Bio-Rad Laboratories, Hercules, California, USA), followed by electroporation at 200 V and 1050 μF with a Gene Pulser Xcell TM electroporation system (Bio-Rad Laboratories) and recultivation in fresh medium.

Techniques: SPR Assay, Binding Assay, Concentration Assay, Standard Deviation, Expressing, Construct, Variant Assay, Electroporation, Plasmid Preparation, Western Blot, FLAG-tag, Solvent, Two Tailed Test, MANN-WHITNEY