Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1-depletion decreases DNA repair by modulating KU70 acetylation levels. ( A ) Jurkat reporter cell lines were generated from parental cell lines by transfection of the pimEJ5-GFP construct and subsequent selection with puromycin for over 14 days. These cell lines were subsequently engineered to lose BCAT1 expression (pLKO.1 sh BCAT1 #3). The reporter cell lines were then electroporated with the pCBA-SceI endonuclease-expressing vector (or empty vector). After 72 h, the activity of the c-NHEJ (pimEJ5-GFP vector-expressing cells) DNA repair pathway was assessed by measuring the percentage of GFP-positive cells using flow cytometry. Error bars indicate ±SD. Results from one of three independent experiments performed with 6–9 replicates are shown. Significance was calculated using an unpaired Mann–Whitney U test. * p < 0.05. ( B ) U2OS cells were engineered to overexpress BCAT1 (BCAT1 myc/DDK). Cells were then transfected with the pimEJ5-GFP vector and pCBA-SceI or empty vector. After 48 h, the activity of the c-NHEJ (pimEJ5–GFP vector-expressing cells) DNA repair pathway was assessed by measuring the percentage of GFP-positive cells using flow cytometry. Error bars indicate ± SD. Results from one of two independent experiments are shown. Significance was calculated using an unpaired Mann-Whitney U test. *** p < 0.001. ( C ) U2OS cells were engineered to overexpress BCAT1 (BCAT1 myc/DDK) or BCAT1 mutants (K222A, SXXS). Cells were then transfected with the pimEJ5-GFP vector and pCBA-SceI or empty vector. After 48 h, the activity of the c-NHEJ (pimEJ5–GFP vector-expressing cells) DNA repair pathway was assessed by measuring the percentage of GFP–positive cells using flow cytometry. Error bars indicate ± SD. Results from one of two independent experiments are shown. Significance was calculated using an unpaired Mann-Whitney U test. * p < 0.05, ** p < 0.01. ( D ) Kinetics of DNA repair in CCRF-CEM control and BCAT1 stable knockdown T-ALL cells (sh BCAT1 #1, sh BCAT1 #2). The number of γH2AX foci (left), 53BP1 foci (middle), and coincident γH2AX/53BP1 foci (right) per nucleus following etoposide treatment are denoted. Each point represents data from a single cell, and the bars denote the median foci number per cell. Top panels: Significance was calculated using the Kruskal-Wallis test. ** p < 0.01, *** p < 0.001. n.s. = not significant. Box–and–whisker plots denote expression from minimum to maximum (bottom). Significance was calculated using an unpaired Mann-Whitney U test. ** p < 0.01, *** p < 0.001. n.s. = not significant. ( E ) CCRF-CEM T-ALL cells (left) were treated with different doses of ERG245 (100–200 µM) for 24 h. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for the indicated proteins. Total KU70 and GADPH are shown as loading controls. Jurkat T-ALL cells (right) were treated with different doses of ERG245 (100–300 µM) or Trichostatin A (TSA; 100 nM) for 24 h. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for the indicated proteins. Total KU70 and GADPH are shown as loading controls. The acetylated KU70/total KU70 protein ratios and γH2AX/GADPH protein ratios are also shown. ( F ) Whole cell lysates from ΔE-NOTCH1 leukemias wild-type and KO for Bcat1 were immunoprecipitated using anti-acetyl-lysine affinity beads or control beads and probed for Ku70 and Bcat1. α-Tubulin is shown as a loading control (input).

Article Snippet: Cell lines with successful modulation of BCAT1 expression were transfected/electroporated with the SceI endonuclease expression vector (pCBA-SceI; Addgene #26477) to induce DSBs.

Techniques: Generated, Transfection, Construct, Selection, Expressing, Plasmid Preparation, Activity Assay, Flow Cytometry, MANN-WHITNEY, Control, Knockdown, Whisker Assay, Western Blot, Immunoprecipitation