Fig. 2. BAG3 is important for removal of nuclear protein during proteotoxic stress.

a, b Western blots show that due to proteasomal inhibition, expression of BAG3 and lamin B was upregulated in the cytosolic fraction as well as in the nuclear fraction. NRVCs were treated with the proteasomal inhibitor MG132 (20 µM) for 6 h and cells were fractionated into cytosolic and nuclear fractions. Expression of BAG3 and lamin B, HSP70, and p62 was determined by western blot in the cytosolic as well as in nuclear fraction by BAG3, lamin B, HSP70, and p62 antibodies, respectively. Expression of cytosolic and nuclear proteins was normalized with GAPDH and lamin A/C expression, respectively. Graph shows the quantification of BAG3, HSP70, p62, and lamin B expression in the cytosol as well as in the nuclear fraction. c, d) Proteasomal inhibition results in the accumulation of ubiquitinated proteins in the nuclear fraction. Cardiomyocytes were treated with MG132 (20 µM) for 6 h, and cytosolic and nuclear fractions were prepared. Western blot was done with the ubiquitin antibody. d Representative images show that inhibition of proteasomal function caused accumulation of micronuclei in the cytosol. Cardiomyocytes were treated with the proteasomal inhibitor for 6 h and fixed with 4% PFA. Fixed cells were stained with the BAG3, lamin B, and nuclei were stained with DAPI. e Representative images show that inhibition of proteasomal function with MG132 causes dysregulation of nuclear shape. MG132-treated cardiomyocytes were fixed with 4% PFA and stained with the lamin B (red). f Representative images show that knockdown of BAG3 or the treatment of the cells with MG132 affects the size and shape of the nuclei and causes accumulation of micronuclei in the cytoplasm. Cardiomyocytes were transduced with the respective adenoviral construct for 48 h and treated with MG132 (20 µM) for 6 h. Cells were fixed with 4% PFA and stained with DAPI. g Quantification of the results (shown in panel f) depicting nuclear area of the control and treated cells (n = 50–100 in each group) as measured by the ImageJ software. h Representative images show the nuclear morphology in wild-type and in BAG3 (+/−) mice heart. i Quantitative presentation of the results shown in panel h. j Examination of lamin B in the cytosolic fractions of NRVCs under the conditions that the level of BAG3 is changed and the cells are under the stress upon the treatment with MG132. Cardiomyocytes were transduced with the respective adenovirus for 48 h and cells were treated with MG132 (20 µM) for 6 h. k Expression of lamin B (under similar conditions as described in j) in nuclear-insoluble fractions. The level of lamin A/C, which was shown to remain unchanged, served as an internal control. NS not significant