Definitive evidence using enucleated cytoplasts for nongenomic effects of STAT5a/b siRNAs on ER structure

STAT5a/b species are well‐known as transcription factors that regulate nuclear gene expression. However, we observed that STAT5a associated with the Golgi apparatus in HPAECs and that siRNA‐mediated knockdown of STAT5a/b led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition along cyst membranes and tubule‐to‐cyst boundaries of reticulon‐4 (RTN4/Nogo‐B), atlastin‐3 (ATL3) and Golgi fragmentation. Moreover, there was marked accumulation of the 63kDa cytoskeleton‐linking membrane protein and ER‐spacer CLIMP63/CKAP4 within the cysts. That the STAT5a/b‐siRNA‐induced cystic ER phenotype developed in the presence of a transcription inhibitor DRB had suggested that the mechanism was nongenomic. We have now definitively tested the requirement for the nucleus in eliciting the STAT5a/b‐siRNA‐induced cystic ER phenotype. Enucleated HPAEC cytoplasts were prepared using adherent 35 mm cultures using the cytochalasin B‐centrifugation method (typically yielding 65–75% enucleation). STAT5a/b siRNAs readily elicited the cystic ER phenotype including the marked luminal accumulation of CLIMP63 and Golgi fragmentation in the recovered HPAEC cytoplasts demonstrably lacking a nucleus. These studies provide unequivocal evidence for a nongenomic mechanism underlying the cystic change in ER structure elicited by STAT5a/b knockdown.