Journal: Nucleic Acids Research

Article Title: A clickable melphalan for monitoring DNA interstrand crosslink accumulation and detecting ICL repair defects in Fanconi anemia patient cells

doi: 10.1093/nar/gkad559

Figure Lengend Snippet: Modification for Click chemistry does not change the activities of click-melphalan or –mono-melphalan. ( A, B ) HeLa cells exposed to various concentrations of the indicated drugs were subjected to clonogenic survival assays. ( C ) Representative images of comet assays conducted on HeLa cells subjected to the indicated treatments. ( D ) Comet assay quantification indicating the decrease of the irradiation-induced comet tail moment with the indicated drugs. ( E ) Detection of FANCD2 and γH2AX in HeLa cell protein extracts after treatment with 1 μM melphalan or click-melphalan. Numbers under the blots indicate the fold induction relative to untreated samples. L/S indicates the ratio of monoubiquitinated (L, upper band) to non-monoubiquitinated (S, lower band) FANCD2. ( F ) Monitoring of cell cycle profile by propidium iodide staining in HeLa cells exposed to melphalan or click-melphalan. Data are shown as mean + s.e.m. of 3 independent experiments. ns (not significant), * P < 0.05, ** P < 0.01 and **** P < 0.0001 according to two-way ANOVA followed by Šidák multiple range test (A, B) or one-way ANOVA non parametric test (D) and Mann–Whitney test (E).

Article Snippet: Cells were stained at room temperature with propidium iodide (PI) solution (2 mM MgCl 2 , 10 mM PIPES buffer, 0.1 M NaCl, 0.1% Triton X-100, 0.01 mg/ml PI) and analyzed on a LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA).

Techniques: Modification, Single Cell Gel Electrophoresis, Irradiation, Staining, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: A clickable melphalan for monitoring DNA interstrand crosslink accumulation and detecting ICL repair defects in Fanconi anemia patient cells

doi: 10.1093/nar/gkad559

Figure Lengend Snippet: Analysis of the FA-dependent melphalan-ICL repair during the cell cycle. ( A ) Dual parameter plot of the click-mono-melphalan or melphalan subjected to the Alexa 647-click reaction with propidium iodide co-staining. Lesions repair is evaluated in HeLa and HeLa FANCD2 KO cells by flow cytometry at different time post treatment. The percentage of G1 phase cells is indicated in the left corner (black), S phase cells in the center (yellow) and G2 phase in the right corner (red). ( B ) HeLa cells were arrested in G1 phase using a double thymidine block. Cultures harvested at various time points after release were stained with propidium iodide and analyzed using flow cytometry. ( C ) Quantification of fluorescence intensity in HeLa WT or HeLa FANCD2 KO treated with click-melphalan clicked with Alexa Fluor 647 azide at the indicated time post treatment. Data are shown as mean + s.e.m. of three independent experiments. * P < 0.05 according to two-way ANOVA followed by Šidák multiple range test.

Article Snippet: Cells were stained at room temperature with propidium iodide (PI) solution (2 mM MgCl 2 , 10 mM PIPES buffer, 0.1 M NaCl, 0.1% Triton X-100, 0.01 mg/ml PI) and analyzed on a LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA).

Techniques: Staining, Flow Cytometry, Blocking Assay, Fluorescence