Fig 2: Analysis of the specificity of commercially available anti-PA200 antibodies. (A) A time course of transient PA200 silencing in human A549 alveolar epithelial cells was analyzed for specific recognition of PA200 by Western blotting using the leading commercially available anti-PA200 antibody #1 (PA1-1961, Thermo Fisher Scientific, Waltham, MA, USA); (B) Protein lysates of testes were prepared from wild type (WT) and PA200-/- (KO) mice (obtained from [7]) and analyzed for detection of PA200 by Western blotting using antibody #1; (C) Immunohistochemistry (IHC) staining of paraffin-embedded testis sections from wild type (WT) and PA200-/- (KO) mice with antibody #1; (D) Samples used in (A) were analyzed with the specific PA200-targeting antibody #2 (NBP1-22236, Novus Biologicals, Littleton, CO, USA); (E) Samples used in (B) were analyzed with the specific PA200-targeting antibody #3 (NBP2-32575, Novus Biologicals, Littleton, USA); (F) Tissue sections from wild type (WT) and PA200-/- (KO) mice were stained with antibody #4 (sc-135512, Santa Cruz, Dallas, TX, USA) by IHC. Figures show representative results for experiments performed with n = 3. (Reproduced with permission from Welk et al., Scientific Reports; published by Nature Publishing Group, 2019 [5]).

Fig 3: PA200 regulates the expression of genes and proteins implicated in the control of cell proliferation and cell stress. The mRNA level of cellular proliferation and stress genes was analysed using quantitative real‐time PCR analyses. Before RNA extraction, shPA200 and control cells were incubated with vehicle (DMSO) or the mitochondrial inhibitors, 10 µmol/L rotenone and 3 µmol/L oligomycin, for 24 h. (A) The mRNA levels of vehicle‐treated shPA200 were normalized to vehicle‐treated controls. (B) The mRNA expression of selective mitochondrial inhibitor‐treated (10 µmol/L rotenone and 3 µmol/L oligomycin) control cells and shPA200 cells was normalized to DMSO‐treated control and DMSO‐treated shPA200 cells, respectively. Puromycin was removed 24 h before performing every experiment. Data are presented as the mean ± SD of four separate experiments. Statistical analysis was performed by ANOVA using GraphPad Prism v8.2.1. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (C) Control and shPA200 neuroblastoma cell lines were incubated with vehicle (DMSO) or 10 µmol/L rotenone for 24 h. Total cell lysates obtained from control and shPA200 cells after 24‐h treatment were used to analyse phosphorylation profiles of kinases using the human phospho‐kinase array. Labels: kinase array panel A: xxx PRAS S40 and xxx CREB; kinase array panel B: xxx WNK 1, xxx c‐Jun and xxx p53. Representative bars of the pixel intensity of dots in the human phospho‐kinase array. For data analyses, Image Lab 5.2.1 software was used. (D) Confirmation of the effects of PA200 on the c‐Jun‐JNK pathway by SDS‐PAGE and Western blot. Control and shPA200 neuroblastoma cell lines were incubated with vehicle (DMSO) or 10 µmol/L rotenone for the indicated time‐points. Total cell lysates (30 µg protein) obtained from control and shPA200 cells at indicated time‐points were separated by SDS‐PAGE, transferred to nitrocellulose membranes and subjected to Western blot analysis. Actin was used as an internal loading control (right panel). Densitometry for c‐Jun and p‐c‐Jun after rotenone treatment was performed on three independent biological replicates of control and shPA200 cells (left panel). Data are presented as mean values ± SD of 3 independent experiments. Statistical analysis was performed by ANOVA using GraphPad Prism v8.2.1. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (E) Activation of the SAPK/JNK pathway was analysed using SDS‐PAGE and Western blot to check total and phosphorylated SAPK/JNK protein levels. Control and shPA200 neuroblastoma cell lines were incubated with vehicle (DMSO) or 10 µmol/L rotenone for the indicated time‐points. Total cell lysates (30 µg protein) obtained from control and shPA200 cells at indicated time‐points were separated by SDS‐PAGE, transferred to nitrocellulose membranes and subjected to Western blot analysis. Actin was used as an internal loading control. The SAPK/JNK antibody detects endogenous levels of total JNK1, JNK2 or JNK3 protein. Western blot analysis was performed in n = 3 independent experiments

Fig 4: PA200 affects cell viability after treatment with mitochondrial inhibitors. PA200‐depleted cells and corresponding control cells were treated with vehicle (DMSO) or mitochondrial inhibitors for 24 h. (A) Cell cytotoxicity was assessed using a sulphorhodamine B assay. Before the assay, 5000 cells were plated in each well of a 96‐well plate. On the following day, cells were treated for 24 h with either DMSO or mitochondrial inhibitors at the indicated concentration in DMEM complete media without puromycin. Results are presented as the mean ± SD of six independent experiments. Statistical analysis was performed by ANOVA using GraphPad Prism v8.2.1. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (B) Assessment of necrosis using the LDH assay. Cells were treated with DMSO, 10 µmol/L rotenone, 3 µmol/L oligomycin or 100 nmol/L antimycin A. Results are presented as the mean ± SD of three independent experiments. Puromycin was removed 24 h before performing every experiment. Statistical analysis was performed by ANOVA using GraphPad Prism v8.2.1. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (C)‐(D) Apoptotic markers were verified by SDS‐PAGE and Western blot in control and shPA200‐depleted cells. Control and shPA200 neuroblastoma cell lines were incubated with vehicle (DMSO), 10 µmol/L rotenone (R), 3 µmol/L oligomycin (O) or 100 nmol/L antimycin A (A) for 24 h. Total cell lysates were separated by SDS‐PAGE, transferred to nitrocellulose membranes and subjected to Western blot analysis to check the level of phosphorylated γH2AX (C) and cleaved PARP1 (D). Actin was used as an internal loading control. Densitometry was performed on three independent biological replicates (C‐D lower panels) with Image Lab software. Data are presented as mean values ± SD and represent 3 independent experiments. Statistical analysis was performed by ANOVA using GraphPad Prism v8.2.1. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001)

Fig 5: Generation of a stable PA200‐depleted neuroblastoma cell line using lentiviral technology. Down‐regulation of the expression of PSME4/PA200 in SH‐SY5Y human neuroblastoma cell line was achieved using lentiviral technology. To produce the virus, HEK293T cells were transfected with a mixture of packaging‐enveloping vectors and the plasmids pGIPZ‐GFP containing the shRNA target sequences (PMSE4 clones #1, #2, #3, #4 and #5). Cells stably expressing empty pGIPZ‐GFP were used as a control. Transduction of SH‐SY5Y neuroblastoma cells was conducted using 8 µg/mL polybrene, and cells were maintained in 1.25 µg/mL puromycin for selection. Puromycin was removed 24 h before performing every experiment. (A)‐(B) PA200‐depleted cells and corresponding control cells were treated with vehicle (DMSO) or mitochondrial inhibitors (10 µmol/L rotenone, 3 µmol/L oligomycin, 100 nmol/L antimycin A) for 24 h. The qPCR analysis showed extensive silencing of PA200 in PMSE4 clone #4 and PMSE4 clone #5 later referred to as shPA200_A and shPA200_B, respectively. Results are presented as the mean ± SD of n = 4 independent experiments. Statistical analysis was performed by ANOVA using GraphPad Prism v8.2.1. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (C) The control cell line was treated with vehicle (DMSO) or mitochondrial inhibitors (10 µM rotenone, 3 µmol/L oligomycin, 100 nmol/L antimycin A) for 24 h. A qPCR analysis was performed to check the relative mRNA expression of PMSE4. Results are presented as the mean ± SD of n = 4 independent experiments. Statistical analysis was performed by ANOVA using GraphPad Prism v8.2.1. (D) Total cell lysates from shPA200 cells (shPA200_A, shPA200_B) and control cells were prepared, and equal protein amounts of each sample were separated by SDS‐PAGE to analyse the protein level of PA200. Actin was used as the loading control