Extended Data Fig. 6: Transcriptionally engaged CycT1 is preferentially targeted for PARylation upon MNNG-induced DNA damage, which inhibits CycT1’s phase separation.
a, & b, CycT1 affinity-purified from whole cell extracts (WCE) of HeLa cells, which were untreated or treated with the indicated chemicals, were analyzed by Western blotting (WB). c, Nuclear extracts (NE) of control or MNNG-treated HeLa cells were subjected to immunoprecipitation with anti-HEXIM1 antibody. The immunoprecipitates (IP) were analyzed by WB. d, The LARP7 protein levels in NE of HeLa cells expressing a LARP7-specific shRNA only in the presence of doxycycline (Dox) were analyzed by WB. e, HeLa cells expressing GFP-CycT1 were untreated (control) or treated with the indicated chemical(s). The distribution patterns of GFP-CycT1 were analyzed by fluorescence microscopy. Scale bar = 10 μM. f, WT and mutant GFP-CycT1-IDR were subjected to in vitro ADP-ribosylation reactions containing NAD+ or not and then analyzed by WB. g, Solutions containing 2 mg/ml of WT or mutant GFP-CycT1-IDR were subjected to droplet formation assay and examined by fluorescence microscopy for GFP signals. Scale bar = 20 μM. Right: Quantification of the sizes of droplets in each group. The box plots show the minimum, first quartile, median, third quartile and maximum with n represents the number of droplets: GFP-CycT1-IDR-WT (n = 849), GFP-CycT1-IDR-Mut2 (n = 621). h, Solutions containing GFP-CycT1-IDR Mut2 proteins that were in vitro PARylated or not were subjected to droplet formation assay and analyzed as in g. Scale bar = 25 μM. Right: Quantification of the sizes of droplets in each group. The box plots are as described in g with n represents the number of droplets: GFP-CycT1-IDR-Mut2-Control (n = 1346), GFP-CycT1-IDR-Mut2-PARylation (n = 1371). i, Liquid droplets containing GFP-CycT1-IDR (0.1 mg/ml) and mCherry-PAR-PARP1 (0.025 mg/ml) were incubated with or without NAD+ and then analyzed by WB. j, Samples analyzed in i were further examined by fluorescence microscopy for GFP and mCherry fluorescence signals. Scale bar = 25 μM. Right: Quantification of the sizes of droplets in each group. The box plots are as described in g with n represents the number of droplets: GFP-CycT1-IDR − NAD+ (n = 1410), GFP-CycT1-IDR + NAD+ (n = 2483); statistical analysis was performed using two-tailed unpaired t-tests; ****P < 0.0001. Experiment in e was repeated independently three times with similar results. g, h, j, Data are representative of three independent experiments with similar results. All Western blots are representative of three independent experiments. Gel source data are available online.
