Bim forms complexes with Bcl2 but not Bax in HS-Csn8KO liver.

<p>Crude proteins extracts from liver tissues were used for immunoprecipitation (IP) followed by iimunoblotting (IB) analyses. (<b>A</b>) Immunoprecipitation using a Bim-specific antibody followed by IB for the indicated proteins. (<b>B</b>) Pooled densitometry data of the IP western blot analyses as illustrated in panel A. For each protein, the density of the Csn8KO group is shown as the relative value to the average value of the corresponding CTL group. Arbitrary unit is used. (<b>C</b>) Immunoprecipitation of Bcl2 was followed by IB for Bcl2, Bim, and Bax. NS denotes a non-specific band. (<b>D</b>) Pooled densitometry data of the IP western blot analyses as illustrated in panel C. (<b>E</b>) Immunoprecipitation of Bax was followed by IB for Bax and Bim. *p<0.05, **p<0.01 vs. CTL.</p

Dynamic effects of ALP inhibition via 3-MA or BFA on p62 protein levels and a surrogate UPS substrate in cultured neonatal rat ventricular myocytes (NRVMs).

<p>Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu, Ad-RFP) 48 hr before 3-methyladenine (3-MA) or bafilomycin A1 (BFA) treatment. <b>A ∼ C</b>, The time course of protein level changes in the GFPu/RFP ratio and the p62/β-tubulin ratio in NRVMs treated with 3-MA (3 mM) or vehicle control. Representative images of western blots (<b>A</b>) and quantitative data from 3 repeats (<b>B, C</b>) are shown. Paired <i>t</i>-test, *<i>p</i><0.01. <b>D</b> and <b>E</b>, 3-MA and BFA concentration-dependently increases GFPu/RFP ratio at 24 hours. At 24 hr after the drug treatment, the cells were harvested for total protein extraction and western blot analyses of the indicated proteins. Representative images (<b>D</b>) and pooled densitometry data (<b>E</b>) are shown. Two-way ANOVA followed by the Scheffé's test; *<i>p</i><0.05, **<i>p</i><0.01 vs. CTL; n = 3 repeats/group.</p

Representative electron micrographs of liver tissue sections.

<p>Liver tissues samples from the CTL (A, C) and HS-Csn8KO (B, D ~ F) littermate mice at 4 weeks of age were processed for transmission electron microscopy. IBL, inclusion-body like structure; Scale bar=1µm.</p

Hepatocytomegaly, nuclear megaly, and increased cytoplasmic inclusions in HS-Csn8KO mouse livers.

<p>(<b>A</b>) Representative images of Csn8 immunohistochemistry (brown). Liver tissues were sampled from HS-Csn8KO and CTL littermate mice at 6 weeks of age. Immunohistochemistry for Csn8 was performed as described in Methods. The nuclei were counter-stained blue with hematoxylin. (<b>B</b>) Representative images of toluidine blue stain of semi-thin sections of resin-embedded liver tissues sampled from HS-Csn8KO and CTL littermate mice at 4 weeks of age. Scale bar=20µm.</p

Lysosomal inhibition accumulates both autophagic and proteasomal substrates in mice: hearts and kidneys.

<p>Male GFPdgn transgenic mice at 10 weeks of age were treated with bortezomib (BZM, 1 mg/kg, i.p.), bafilomycin A1 (BFA, 2.5 mg/kg/12 hrs, i.p.), or vehicle control (CTL). The mice were sacrificed at either 3 or 24 hours after the first injection and myocardial and kidney tissues were collected for total protein and RNA extraction and subsequent analysis. <b>A</b> and <b>B</b>, Representative images (A) and pooled of densitometry data (B) of western blot analyses for myocardial GFPdgn and other indicated proteins. <b>C</b>, Representative images (upper) and pooled densitometry data (lower) of western blot analyses for renal GFPdgn protein levels. <b>D</b> and <b>E</b>, Representative images (upper) and pooled densitometry data of reverse transcription (RT) PCR analyses of GFPdgn mRNA levels in heart and kidney tissues. Total RNA was used for the RT to synthesize the first strand cDNA which was subsequently used for GFPdgn and GAPDH duplex RT-PCR. GAPDH was probed as loading control. N = 4 mice/group. *<i>p</i><0.05, **<i>p</i><0.01 vs. CTL.</p

HR-Csn8KO impairs UPS proteolytic function in mouse livers.

<p>CTL::GFPdgn tg and H-Csn8KO::GFPdgn tg littermate mice resulting from cross-breeding between GFPdgn tg and HS-Csn8KO mice were used at 4 weeks of age. (<b>A</b>, <b>B</b>) Representative image (A) and pooled densitometry data (B) of western blot analyses of hepatic GFPdgn. A nonspecific band at a molecular weight of ~80kDa is used as the loading control (LC). (C) Representative fluorescence confocal micrographs of GFPdgn direct fluorescence. Scale bar=50 µm. (D, E) Western blot analyses of Rpt5 in this cohort of mice. *p<0.01 vs. CTL::GFPdgn, n=4 mice/group; Student’s t-test.</p

Temporal characterization of hepatocyte apoptosis in HS-Csn8KO mice.

<p>(A) Representative images of TUNEL staining of liver cryosections from 6-week-old mice. Scale bar: 50µm. (B) The time course of changes in TUNEL positivity in the liver during postnatal 6 weeks. Four livers each group and 300 nuclei per liver were counted. #p<0.05, *p<0.01 vs. CTL.</p

Representative confocal micrographs of mouse myocardial direct green fluorescence.

<p>Adult GFPdgn non-transgenic (<b>A</b>) and transgenic (<b>B</b> ∼ <b>D</b>) mice at 10 weeks of age were treated with bortezomib (1 mg/kg, i.p.; <b>C</b>), bafilomycin A1 (BFA, 2.5 mg/kg/12 hrs, i.p.; <b>D</b>), or vehicle control (DMSO, <b>A</b> and <b>B</b>). At 24 hours after the first BFA injection, ventricular myocardial tissues were collected and immediately immersion-fixed with 3.8% paraformaldehyde and processed for cryo-sectioning. The cryosections were mounted and imaged for direct fluorescence via confocal microscopy. Scale bar = 100 µm.</p

HS-Csn8KO yields differential effects of on proteasome subcomplexes in mouse livers.

<p>(<b>A</b>) Representative images of western blot analyses of the indicated subunits of the 19 and the 20S proteasome in the liver of 4-week-old mice. (<b>B</b>) Pooled densitometry data of the western blot analysis of Rpt5 as illustrated in panel A. (<b>C</b>, <b>D</b>) HS-Csn8KO resulted in the upregulation of three subunits of the 11S proteasome. β-Tubulin was probed as the loading control. AU denotes arbitrary unit. n=4 mice/group; #p<0.05, *p<0.01 vs. CTL; Student’s <i>t</i>-test.</p

Increased protein expression of Bcl2 family members in HS-Csn8KO mouse livers.

<p>Four pairs of HS-Csn8KO and CTL littermate mice at 4-week-old were used for analyses presented here. (<b>A</b>) Representative images of western blot analyses of the indicated Bcl2 family proteins. β-Tubulin was probed as loading control (B~D). Summary of quantitative analyses. Pooled densitometry data of Bim (<b>B</b>), Bax (<b>C</b>), Bcl2 and Bcl-xL (<b>D</b>) normalized to β-tubulin are presented. *p<0.05, **p<0.01 vs. CTL; n=4 mice/group.</p