Histone deacetylation regulates nucleotide excision repair through an interaction with the XPC protein

Fluorescence-microscopy-based assay assessing regulatory mechanisms of global genome nucleotide excision repair in cultured cells

“… After obtaining the surviving transformants, confirm the stable expression of EGFP-XPC by western blot analysis according to the previously described procedure ( Figure 1 A). 1

Figure 1 Local UVC irradiation through a polycarbonate isopore membrane filter (A) Immunoblot analysis confirming the expression of XPC and DDB2 proteins in the cell lines used. For U2OS DDB2 KO cells, the presence of biallelic mutations of the endogenous DDB2 gene was confirmed by sequencing; therefore, the faint band marked by an asterisk represents a nonspecific reaction of the antibody used.

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“… 6 , 7 Our recent studies also took advantage of this technique and proposed that histone deacetylation is introduced at DNA damage sites and supports damage recognition by XPC. 1 , 8 To allow statistical evaluation of the results from the subnuclear DNA damaging experiments in our studies, it was crucial to quantitatively measure fluorescence signals (either fluorescent protein tag or immunofluorescence) at local UV damage (LUD) sites for various chromatin-related protein factors and/or histone modifications. Herewith we describe our detailed protocols, which are applicable for assessing the localization of various factors upon local UV irradiation in chromatin (we also utilize laser-based local UV stimulation based on three-photon absorption, 9 , 10 which is not included because it requires a customized, expensive equipment and is not widely available).…”

“… Note: This procedure can be used to establish stable transformants of various cultured cells other than U2OS, such as XP patient-derived cell lines and mouse embryonic fibroblasts. 1 , 12 Alternatives: Other plasmid- or virus-based expression systems are also applicable for establishing cells stably expressing XPC fused to a fluorescent protein. …”

“…Note: This procedure can be used to establish stable transformants of various cultured cells other than U2OS, such as XP patient-derived cell lines and mouse embryonic fibroblasts. 1 , 12 …”

“… 13 N/A U2OS WT cells stably expressing FLAG-EGFP-XPC Kusakabe et al. 1 N/A U2OS DDB2 KO cells stably expressing FLAG-EGFP-XPC Kusakabe et al. 1 N/A 293-GPG Ory et al.…”