Fig. 1.

PARP1 is crucial for inflammatory stimulation-induced HuR oligomerization in cells. a PARylation is required for the stimulation-dependent increase in HuR oligomerization shown by in situ chemical crosslinking. HEK293 cells were either exposed to TNFα (± Ola) for 1 h or not, and then the cells receiving different treatments were suspended independently in PBS and incubated with or without 1 mM DSS. After quenching the reaction using a final concentration of 20 mM Tris at room temperature for 15 min, the extracts were lysed with RIPA buffer, followed by western blotting with an anti-HuR antibody. b HuR undergoes self-associate in cells responding to inflammatory stimuli. HEK293 cells were transfected with the Flag-HuR plasmid and treated with or without TNFα (± Ola). Cells extracts were immunoprecipitated with control IgG1 or an anti-FLAG antibody, and the precipitants were analyzed by western blotting with an anti-HuR antibody. Endo HuR, endogenous HuR. c, d Oligomerization of HuR and the mutants were analyzed using a PLA. GFP-HuR together with Flag-HuR or mutated plasmids, as indicated, was transfected into cells. The cells were mock-treated or TNFα-exposed (± Ola) for 1 h, and then subjected to in vivo PLA assays with indicated antibodies. Scale bar, 10 μm (c). The quantitative results (d) are represented from three independent experiments. ***p < 0.001. e PARP1 knockdown impairs HuR oligomerization. HEK293 cells were transfected with PARP1 siRNA or a control, and then, the Flag-HuR expression plasmid was transfected independently. After 48 h, the differently treated cells were exposed to TNFα for 1 h, and then, immunoprecipitates were prepared using a FLAG antibody. They were subjected to western blotting to detect the interaction of Flag-HuR with the endogenous HuR. Endo HuR, endogenous HuR. f The HuR D226 mutation decreases its oligomerization in response to TNFα exposure. HEK 293 cells were transfected independently with wild-type (WT) Flag-HuR, W261E and D226A mutant plasmids, and then either challenged with TNFα or not for 1 h. Immunoprecipitates were prepared using control IgG1 or an anti-FLAG antibody and then subjected to western blotting to detect the interaction of Flag-HuR with the endogenous HuR. Endo HuR, endogenous HuR