Fig. 9: FAM122A is localized in the nucleus in interphase and to DNA damage foci in response to replication stress.

A mRFP-FAM122A wild-type was co-transfected with GFP-h-H2B into HEK293T cells and observed by time-lapse confocal microscopy (20x objective, 2.5x zoom factor). Arrows indicate the cell(s) in the cell cycle phase. B mRFP-FAM122A, BFP-B55α and GFP-h-H2B expressing vectors were transfected into HEK293T cells to monitor localization. Close up of two cells in interphase (I) and in Mitosis (M) are shown (the nuclear envelope is traced with a dashed white line to demonstrate expression of B55α in both the cytoplasm and the nucleus). C U-2 OS cells were transfected B55α and FAM122A SplitFast constructs and colocalization was visualized by adding the HMBR fluorogen to the media 48 h later. Experiment performed as biological triplicate. D FAM122A KO and reconstituted WT expressing C1 HEK293 cells were treated with 2 mM HU for 24 h, stripped of cytoplasmic proteins and fixed for immunofluorescence imaging to observe localization of FAM122A bound to chromatin in the context of γH2AX foci on a confocal microscope using a 63x objective and 5x zoom factor. The colocalization of FAM122A with γH2AX foci are represented by the yellow signal. Experiment performed as biological triplicate. E FAM122A KO and FAM122A reconstituted HEK293 cells (WT and SLiM MT) were treated with 2 mM HU for 3 h and subjected to gamma γH2AX immunofluorescence where mean-fluorescent intensity was analyzed using a Kruskal-Wallis test (two-sided) with a Dunn’s multiple comparison test. KO:WT Recon. p-value = 0.0429. KO:SLiM MT Recon. p-value = 0.0141. n = 3 biological replicates. Source data are provided as a Source Data file.