Fig. 5

PSMD11 could be conditionally depleted in vitro and in vivo and could induce massive apoptosis in MEFs. a Genetic strategy to knockout PSMD11 by tamoxifen-mediated CreER^T2 activation. b Genotyping of DNA from E14.5 embryos from intercrossing between Flp;FSF-R26^{CAG − CreERT2/+};PSMD11 ^flx/+ mice. Sizes of WT and mutant PCR bands are indicated. c-e MEFs were isolated by a standard protocol from E14.5 embryos with the genotype of Flp;FSF-R26^{CAG − CreERT2/+};PSMD11^flx/flx, then the cells were treated for 5 days with 0.5uM 4-hydroxytamoxifen or vehicle (ethanol) to induce knockout of PSMD11, then Genotyping (c), immunofluorescence (d) and qRT-PCR (e) were performed to detect the change of expression of PSMD11. Control DNA was from a nonrecombined PSMD11 ^flx/+ mouse without Cre expression, nuclei are counterstained with DAPI (blue) in immunofluorescence staining. Scale bars, 50 μm. f MEFs were treated with 0.5uM 4-hydroxytamoxifen or vehicle (ethanol) for the indicated times, then Western blotting was performed with antibodies directed against PSMD11, PSMD4, PARP, cleaved-Caspase 3, Ubiquitin, β-actin was used as loading control. g After treated with 0.5uM 4-hydroxytamoxifen or vehicle (ethanol) for 5 days, MEFs were stained with 1 μg/mL Hoechst 33342 for 20 min and imaged by confocal laser-scanning microscopy. Arrows denote apoptotic cells. h The chymotrypsin-like activity of proteasome was tested in 30 μg whole-cell extracts from vehicle or tamoxifen treated fibroblasts for 4 days with 0.1 mM Suc-LLVY-AMC peptide at 37 °C for 180 min in quadruplicates on a 96-well plate, Y-axis indicated the relative fluorescence units (RFU) reflecting the AMC cleavage from the peptide. Bars showed the mean ± SD. i The FCP ^flx/flx and WT mice were fed with tamoxifen-containing food (400 mg/kg) or vehicle for 3 days, then the pancreas, liver and kidney were collected to detect the expression of PSMD11 and P53 with β-actin as loading control with Western blotting