Fig 2: Mitochondrial defect increases NFE2L1 transcription via reactive oxygen species (ROS). (A, B) Mitochondrial ROS (A) and cellular ROS (B) levels were monitored by flowcytometric analysis after staining cells with MitoSOX and DCFH-DA, respectively. ** p < 0.01 vs. Ch-L by the Student t-test. (C–F). Ch-L was treated with the indicated concentrations of H2O2 for the indicated time periods. ** p < 0.01 vs. C by the Student t-test. (C) Cellular ROS level was monitored by using DCFH-DA (left panel), and the NFE2L1 mRNA level was examined by qRT-PCR (right). (D) Western blot. (Figure S2) SNU423 was used as a control of NFE2L1 level. (E) Immunocytochemistry for the intracellular localization of NFE2L1. Nucleus was stained with DAPI. NFE2L1 overexpressed by the transfection of NFE2L1-V5 plasmid was used as a control. (F) Time-course NFE2L1 expression in response to H2O2. NFE2L1 mRNA level by qRT-PCR (left) and cellular ROS level (right). (G) SNU387 was treated with 50 μM H2O2 for the indicated time periods. Lower H2O2 concentrations were applied to SNU387 due to the cell’s different sensitivity to H2O2 from Ch-L. Over 100 μM H2O2 induced the cell death of SNU387. NFE2L1 mRNA level (left panel) and cellular ROS level (right panel). ** p < 0.01 vs. C by the Student t-test. (H,I) SNU423 was treated with 20 mM N-acetyl cysteine (NAC) for 24 h. (H) Western blot. (Figure S2) (I) Cellular ROS level. (J) Schematic design of NFE2L1-pGL3 reporter plasmid containing NFE2L1 promoter region from −1580 to +318. (K) Ch-L was transfected with NFE2L1-pGL3 plasmid together with renilla luciferase plasmid for 48 h and then exposed to 400 μM TTFA (a complex II inhibitor) for 12 h. Promoter activity (left) and cellular ROS level (right). (L,M) SNU423 was transfected with NFE2L1-pGL3 plasmid and renilla luciferase plasmid for 48 h and then treated with 20 mM NAC (L) for 24 h or with the indicated concentrations of H2O2 for 48 h (M). Promoter activity (left) and cellular ROS level (right) were monitored. ** p < 0.01 vs. C by the Student t-test.

Fig 3: Ddi2 binds large ubiquitylated proteins via its UBL domain.A, deletion of Ddi2 leads to an accumulation of Ub conjugates, especially large (slowly migrating) conjugates. Immunoblots of WT or Ddi2 knockout HAP1 cells treated with 100 nM bortezomib (BTZ) for 16 h in cells stably expressing GFP (-) or shRNA to Nrf1. Molecular weights here, and throughout, are shown as kilodalton based on the apparent migration size of BLUEstain Protein Ladder (Gold Biotechnology, Inc). B, Ddi2 preferentially binds large Ub conjugates. About 20% input, FLAG-IP, and unbound fractions from HEK293A cells transfected with FLAG-Ddi2 (+) or with an empty vector (–). C, knockout of Ddi2 leads to increased K11/K48 branched chains. HAP1 cells were treated with 5 µM of the p97 inhibitor NMS-873 (873), 1 µM of the proteasome inhibitor BTZ, or 10 µM of the Hsp90 inhibitor pifithrin µ (p µ) for 5 h. A representative image of three blot separate experiments is shown. D, Ddi2, like Rad23B, binds preferentially to large Ub conjugates. Lysates from HAP1 Ddi2 knockout cells treated with 100 nM BTZ for 16 h (to cause accumulation of Ub conjugates) or 5 µM TAK-243 (E1i) for 2 h (to eliminate ubiquitylated proteins) were run on SDS-PAGE gels. Membranes were probed with recombinant Ddi2 or Rad23B protein and blotted with antibodies against Rad23B, Ddi2, or Ub (P4D1). Asterisks denote likely crossreaction with endogenous Ddi2 (*) or Rad23B (**). A representative of two independent experiments is shown. E, the Ddi2 UBL domain is necessary for binding Ub conjugates. Immunoblots of FLAG affinity pulldown from lysates of HEK293A cells expressing full-length FLAG-Ddi2 (WT), ?UBL truncation, ?UIM truncation, or the double deletion of UBL and UIM (??).HEK293A, human embryonic kidney 293A cell line; IP, immunoprecipitation; Ub, ubiquitin; UBL, Ub-like domain; UIM, Ub-interacting motif.

Fig 4: Involvement of MT2/Bip/NRF1 pathway in melatonin-induced ABCA1 expression. a and b Cholesterol (200 μM, 24 h) was treated to UCB-MSCs and the changes in expression levels of MT1 and MT2 were measured by qPCR or western blotting (n = 5, *p < 0.05 vs control). c Melatonin (1 μM) was pretreated to UCB-MSCs before cholesterol treatment (200 μM, 24 h) and then the expression levels of HSPA5, DERLIN, HERP, SEL1L, and VCP were compared by qPCR (n = 5, *p < 0.05 vs cholesterol). d MT2 inhibitor, 4-P-PDOT (10 μM) were pretreated prior to melatonin treatment (1 μM) and the expression level of BiP was measured by western blotting (n = 5, *p < 0.05 vs control, #p < 0.05 vs cholesterol + melatonin). e Immunocytochemistry was conducted with NRF1 (green) and BiP (red) specific antibodies and DAPI (blue) and then visualized by SRRF imaging system. Scale bar was set as 8 μm (magnification × 1000) (n = 5, *p < 0.05 vs control, #p < 0.05 vs cholesterol). f, g NRF1 or NT siRNA was transfected to UCB-MSCs using transfection reagents prior to cholesterol treatment (200 μM, 24 h). The expression levels of ABCA1 and NRF1 were measured by western blotting and the level of total cholesterol was quantified with cholesterol quantification kit (n = 5, *p < 0.05 vs NT siRNA, #p < 0.05 vs NT siRNA + cholesterol). All blot and images are representative and quantitative data are presented as a mean ± standard error of the mean

Fig 5: Non-cleavable Nrf1 mutant is functionally defective.(A) Nrf1-/- MEFs were transfected with constructs expressing either Nrf13×Flag, Nrf1(m1)3×Flag, or vector control and 48 hr later were subjected to Geneticin selection. These antibiotic resistant cells were treated with 1 µM MG132 for 10 hr as indicated. Lysates from these cells were immunoblotted with anti-Flag antibodies to detect exogenous Nrf1. ß-actin was used as a loading control. (B) RNA from Nrf1-/- MEFs transfected and selected as described above was used for quantitative RT-PCR to assess the mRNA levels of representative PSM genes. The values were normalized to GAPDH mRNA levels. Error bars denote SD (n = 3).DOI: http://dx.doi.org/10.7554/eLife.01856.004