Fig 2: Psmb8 promotes Drp1 degradation.A Lysates from siRNA-control- or siRNA-Psmb8-infected NRCMs following H/R 24 h were IP with anti-Drp1 antibody and then analyzed with immunoblottong with anti-ubiquitin (Ub) or anti-Drp1 antibody (left). Input for each protein (right). Quantification of ubiquitinated Drp1 level (right, n = 3). B Lysates from NRCMs infected with Ad-GFP or Ad-Psmb8 and then following H/R 24 h were IP and then analyzed as described in (A). C WT and Psmb8-KO mice were subjected to sham or I/R surgery for 24 h. Heart lysates were immunoprecipitated with an anti-Drp1 antibody. Immunoblotting analysis of ubiquitin-conjugated Drp1 (left). Quantification of the relative ubiquitinated Drp1 protein level (middle, n = 3), and input of Psmb8 and Drp1 proteins (right). D rAAV9-Psmb8 or rAAV9-GFP-injected mice were subjected to sham or I/R surgery for 24 h. Heart lysates were immunoprecipitated and immunoblotting analysis of ubiquitin-conjugated Drp1 (left) as well as input (left) were performed as in (C). E NRCMs were first infected with adenovirus expression siRNA-control or siRNA-Psmb8 for 24 and then stimulated with CHX (10 μM) for another 6, 12 or 24 h. The protein levels of Drp1 and Psmb8 for each time point were determined by immunoblotting analysis with anti-Drp1 or anti-Psmb8 antibody (left) and quantification of the ubiquitinated Drp1 level (right; n = 3). F NRCMs were infected with Ad-GFP or Ad-Psmb8 and treated and analyzed as described in (E). All values are expressed as the mean ± SEM, and n indicates the number of independent experiments or samples per group.

Fig 3: Cytotoxicity of FA and replication stressors in H460 cells. (A) Westerns for LMP2 and LMP7 in cells treated with FA and ONX-0914 (LMP7-i) for 3 h. (B) Westerns for cells treated with LMP7-i and MG132 (5 μM) for 4 h. (C) FACS profiles of EdU incorporation in cells treated with LMP7-i for 6 h. EdU was added during the last hour. (D) Clonogenic survival of cells treated with LMP7-i for 24 h. (E) Clonogenic survival of cells cotreated with FA and 0.5 μM LMP7-i for 3 h and then incubated for 24 h with 0.5 μM LMP7-i (means ± SD for three experiments in triplicates, *p < 0.05, **p < 0.01, ***p < 0.001). (F) Clonogenic survival of cells cotreated for 24 h with 0.5 μM LMP7-i and hydroxyurea or (F) camptothecin (means ± SD for two experiments in triplicates).

Fig 4: Genotoxic signaling in H460 cells. Cells were treated with FA for 3 h except for panel D. LMP7-i (0.5 μM) was present during and after FA treatments (p-CHK2, p-CHK1, p-KAP1, p-p53: phosphorylated forms). (A) Genotoxic signaling at 0 h or (B) 4 h recovery after FA exposure. (C) Westerns for markers of p53 activation at 4 h recovery post-FA. (D) Phosphorylation of CHK2 and p53 in cells treated with FA for 1 or 2 h. (E) Percentage of cells containing γ–H2AX foci (γ–H2AX+) and both γ–H2AX foci and EdU staining (γ–H2AX+/EdU+) (means ± SD, n = 3). (F) As panel E except that cells were fixed at 3 h post-FA exposure (means ± SD, n = 3).

Fig 5: Overexpression of Psmb8 reduces H/R-stimulated cardiomyocyte apoptosis, ROS level and mitochondrial fission in vitro.A Neonatal rat cardiomyocytes (NRCMs) were infected with adenovirus expressing Ad-GFP or Ad-Psmb8 for 24 h and then simulated with H/R for another 24 h. Immunostaining of NRCMs with TUNEL (red) and DAPI (blue) to detect apoptotic cardiomyocytes (left). Quantification of the percentages of TUNEL-positive nuclei (right, n = 3 independent experiments). Bar: 50 μm. B Immunostaining of mitochondrial morphology was performed in cardiomyocytes using MitoTracker® Red (left). Quantification of fragmented mitochondria (right, n = 3 independent experiments). Bar: 10 μm. C NRCMs were infected with adenovirus expressing siRNA-control or siRNA-Psmb8 for 24 h and then stimulated with H/R for another 24 h. Immunostaining of NRCMs with TUNEL (red) and DAPI (blue) was performed to detect apoptotic cardiomyocytes (left). Quantification of the percentages of TUNEL-positive nuclei (right, n = 3 independent experiments). Bar: 50 μm. D Immunostaining of mitochondrial morphology in cardiomyocytes with MitoTracker® Red (left) and analysis of fragmented mitochondria (right, n = 3 independent experiments). Bar: 10 μm. E Detection of the Psmb8, Drp1, p-Drp1(S616), Mfn1, and Mfn2 protein levels with immunoblotting (left) and analysis of each protein level (right, n = 4 each group). F Detection of the Psmb8, Drp1, p-Drp1 (S616), Mfn1, Mfn2, Mito-Drp1 and Mito-Drp1 (S616) protein levels with immunoblotting (left) and analysis of each protein level (right, n = 4 each group). All values are expressed as the mean ± SEM, and n indicates the sample numbers per group.