Visualizing Psoralen-Induced DNA Crosslinks

To generate localized psoralen-induced intrastrand crosslinks and ICLs, U2OS and YFP-CSB^del expressing CS1AN cells were grown on 24 mm coverslips and treated with 50 μM of 8-MOP for 2 h. A 355-nm UV-A laser, set at 24% intensity laser power and low (8%) speed, attached to a PALM laser dissection microscope (Zeiss) equipped with a 40× 0.60 NA Korr LD Plan Neofluar objective, was used to activate 8-MOP in a track along cell nuclei. Irradiated cells were fixed with 2% paraformaldehyde supplemented with 0.1% Triton X-100 and washed in PBS buffer (PBS containing 0.15% glycine and 0.5% BSA) after which immunofluorescence was performed as described (28 (link)). Antibodies used were against CSB (E-18, 1:250, Santa Cruz, sc-10459), FANCD2 (FI-17, 1:1000, Novus Biologicals, nb100-316), XPA (FL-273, 1:100, Santa Cruz, sc-853), γH2AX (JBW301, 1:1000, Millipore, 05-636). Secondary antibodies were conjugated with Alexa Fluor 488, 555 and 633 (Invitrogen). DAPI vectashield (Vector Laboratories) was used to mount coverslips. Slides were imaged using an LSM700 confocal microscope (Zeiss).

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Slyskova J., Sabatella M., Ribeiro-Silva C., Stok C., Theil A.F., Vermeulen W, & Lans H. (2018). Base and nucleotide excision repair facilitate resolution of platinum drugs-induced transcription blockage. Nucleic Acids Research, 46(18), 9537-9549.

Publication 2018

PALM laser dissection microscope FL-273 JBW301 Alexa Fluor 488 DAPI vectashield LSM700 confocal microscope

8 mop Alexa fluor 488 Antibodies Biologicals Buffer Cell nuclei Cells Confocal microscope Dapi Dissection Fancd2 Glycine Immunofluorescence Laser microscope Novus Palm Paraformaldehyde Psoralen Triton x 100 Vector

Corresponding Organization : Erasmus MC

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Variable analysis

independent variables

Treatment with 50 μM of 8-MOP for 2 h
Activation of 8-MOP in a track along cell nuclei using a 355-nm UV-A laser, set at 24% intensity laser power and low (8%) speed

dependent variables

Presence of psoralen-induced intrastrand crosslinks and ICLs
Localization of CSB, FANCD2, XPA, and γH2AX proteins

control variables

U2OS and YFP-CSB^del^ expressing CS1AN cells grown on 24 mm coverslips
Fixation of irradiated cells with 2% paraformaldehyde supplemented with 0.1% Triton X-100
Washing of fixed cells in PBS buffer (PBS containing 0.15% glycine and 0.5% BSA)
Use of specific primary antibodies (anti-CSB, anti-FANCD2, anti-XPA, anti-γH2AX) and secondary antibodies conjugated with Alexa Fluor 488, 555, and 633
Mounting of coverslips using DAPI vectashield
Imaging of slides using an LSM700 confocal microscope

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