Fig. 3

ZMYND11 physically interacts with HNRNPA1 in vitro and in vivo and inhibits HNRNPA1-mediated formation of stress granules. a Venn diagram showing the overlap between three replicates of the identified ZMYND11-interaction proteins in LNCaP cells using co-immunoprecipitation coupled to mass spectrometry (Co-IP/MS). b Table showing top-scoring ZMYND11-interaction proteins reproducibly identified by Co-IP/MS as indicated in (a). c Immunoprecipitation (IP)-Western blot for analyzing the interaction between ZMYND11 and HNRNPA1 in HEK293T cells cotransfected with the indicated V5- or T7-tagged plasmids. IB immunoblot. d Immunoprecipitation-Western blot analysis of an endogenous interaction of ZMYND11 with HNRNPA1 in 22Rv1 cells. IgG immunoglobulin, IB immunoblot. e Immunofluorescence staining and colocalization analysis of ZMYND11 and HNRNPA1 in 22Rv1 cells. Scale bar, 25 μm. f Schematic illustration of the full-length ZMYND11 and the indicated domain deletion mutants. g Immunoprecipitation-Western blot analysis of the interactions between wild type ZMYND11 or its mutants and HNRNPA1. Crude total cell lysates were extracted from HEK293T cells co-expressing T7-tagged HNRNPA1 and the indicated V5-tagged ZMYND11 variants. h 22Rv1 cells stably expressing control or shRNAs against ZMYND11, treated with sodium arsenite (SA, 0.5 mM) for 1 h followed by immunostaining analysis of stress granule (SG) formation. Scale bar, 25 μm. i The number of SGs per cell and ZMYND11 or HNRNPA1 colocalized SGs each cell were quantified. **p < 0.01, ***p < 0.001 evaluated by two-tailed Student’s t test. j Immunoblotting analysis of ZMYND11 or HNRNPA1 in the nuclear and cytoplasmic fractions of the tested prostate cancer cell lines 22Rv1 and LNCaP, respectively. k Apoptosis of 22Rv1 cells stably expressing control or shRNAs targeting ZMYND11. ***P < 0.001 assessed by two-tailed Student’s t test