Supplementary Material for: Knockdown of PSMB7 Induces Autophagy in Cardiomyocyte Cultures: Possible Role in Endoplasmic Reticulum Stress

Proteasomal and autophagic pathways of protein degradation are two essential, endoplasmic reticulum (ER)-associated proteolytic systems involved in the ER stress response. The functional interaction between them has been shown by proteasome pharmacological inhibition. However, little data have been found concerning autophagy induction using an RNA interference approach due to the multisubunit composition of proteolytic systems. We suggested that silencing of single proteasome subunits can induce massive autophagy. <i>Psmb7</i>-specific small interference RNA added to isolated cardiomyocytes significantly affected mRNA expression of essential ER stress marker proteins, including DDIT3/CHOP and HSPA5/GRP78. mRNA expression of the key autophagy regulator MTOR was also increased. These findings were confirmed by single-cell reverse transcription real-time PCR on individual monodansylcadaverine (MDC)-labeled cardiomyocytes. RNA interference that decreased the levels of non-catalytic PSMB7 subunits of the proteasome had no influence on chymotrypsin- and trypsin-like activities, but significantly decreased peptidyl-glutamyl peptide-hydrolyzing activity. Immunohistochemical analysis showed increased levels of LC3-marked vacuoles in the cytoplasm of <i>Psmb7</i>-knockdown cells, and MDC staining showed significantly increased numbers of neonatal cardiomyocytes with autophagic vacuoles. After anoxia-reoxygenation, the number of cells with signs of autophagy after <i>Psmb7</i> gene silencing was higher. Our results indicate that <i>Psmb7</i> knockdown induces ER stress and autophagy in cardiomyocytes, which may be a useful approach to activate specific autophagy.