Matrix and IMS stress trigger distinct UPR responses. (A) Equal amounts of mitochondria isolated from MCF-7 cells transfected with plasmids encoding mutant EndoG-N174A (Endo G) conjugated to GFP for 24 hours were assayed before and after proteinase K treatment in the presence or absence of Triton X-100. Levels of cytochrome c, EndoG-N174A and BCL-XL were detected by immunoblotting (IB) with the indicated antibodies. (B) Lysates of cells transfected with the plasmids encoding the indicated proteins (WT-Endo G, wild-type EndoG–GFP; N-174A, EndoG-N174A–GFP) were subjected to immunoprecipitation (IP) to pull down PHB2 by use of the anti-FLAG antibody. Levels of EndoG in complex with PHB2 or in crude extracts were detected with the anti-GFP antibody. (C,D) Crude extracts from MCF-7 cells transfected with the indicated plasmids (CHOP-Reporter, CHOP promoter driving GFP; Vector, GFP control vector) for 24 hours were subjected to immunoblotting to detect the levels of either GFP (27 kDa) driven by the CHOP promoter (C, top panel) or endogenous CHOP (D) by using anti-GFP or anti-CHOP antibodies, respectively. Levels of EndoG–GFP (57 kDa, Endo G) and tubulin (bottom panel) were detected by using anti-GFP and anti-tubulin antibodies, respectively. Levels of OTC and OTCΔ in the mitochondrial fraction (middle panel) were monitored using an anti-OTC antibody. Thapsigargin (Tg; 1.5 μM) was used as a positive control. The numbers in C represent the fold induction of GFP protein levels normalized to tubulin in cells transfected with EndoG-N174A, OTC and OTCΔ relative to control cells transfected with the CHOP reporter and the control GFP vector. The numbers in D represent the fold induction of CHOP expression levels normalized to tubulin in cells transfected with EndoG-N174A, OTC, OTCΔ and Tg relative to control cells transfected with GFP vector (Vector). (E) Levels of endogenous EndoG mRNA were quantified by qRT-PCR in cells transfected with the indicated plasmids (Vector, control GFP; WT Cyto C, wild-type cytochrome c; WT Smac, wild-type Smac). Data are the means+s.d. of the fold increase relative to control cells overexpressing control GFP from three independent experiments performed in triplicate. (F) Crude extracts from cells overexpressing the indicated plasmids were analyzed by immunoblotting to determine the levels of BiP (BIP) and BIM using anti-BiP and anti-BIM antibodies, respectively. Thapsigargin (1.5 μM) was used as a positive control. (G) Cells maintained in phenol-free medium and transfected with the indicated plasmids were assayed after 24 hours for ER transcription activity by measuring the ERE-driven luciferase activity. Data are means+s.d. of the fold increase relative to the control cells overexpressing control GFP from five independent experiments performed in triplicate. (H) Levels of the indicated IMS proteins were determined in cells overexpressing the respective plasmids by immunoblotting using an anti-GFP antibody. (I) Transiently transfected cells overexpressing either mitochondrially targeted wild-type (WT SOD1) only or mutant SOD1 (Mut SOD) with control GFP were analyzed for ER activity by mean of the ERE-driven luciferase assay. Data are the mean+s.d. of the fold increase relative to the control cells overexpressing GFP from three independent experiments performed in triplicate. Levels of SOD1 and GFP were evaluated by immunoblotting using anti-GFP antibody. Tubulin served as the loading control. *P<0.05; **P<0.01; ***P<0.001 for the elevation in ERE luciferase activity compared with that observed in control vector-treated cells.

ROS-mediated AKT activation is required to induce ERα activity. (A) Cells transfected with either scrambled siRNA (Ctr), or siRNA against ERα or ERβ (10 nM), were incubated in phenol-free medium for 24 hours followed by co-transfection with plasmids encoding mutant EndoG-N174A–GFP (N-174A), the ERE reporter and PRL, which encodes a constitutive luciferase reporter emitting a different wavelength and is used as a loading control. A luciferase assay was performed 24 hours later. Data are the means+s.d. of the fold increase relative to control cells overexpressing control GFP from three independent experiments performed in triplicate. ***P<0.001. (B) Lysates of cells transfected with scrambled siRNA, or siRNA against ERα or ERβ, were collected 5 days after transfection and analyzed by western blotting to evaluate the efficiency of ERα– or ERβ–knockdown using an anti-ERα or anti-ERβ antibody. (C) Subcellular fractionation of cells overexpressing either GFP (Vector) or N-174A were prepared as described in the Materials and Methods section and analyzed by western blotting using the anti-ERα antibody. Lamin A/C, Hsp90 and VDAC represent nuclear, cytoplasmic and mitochondrial protein loading controls, respectively. (D) Crude extracts of the cells transfected with the indicated plasmids (WT-Endo G, wild-type EndoG–GFP) were analyzed by western blotting using an antibody that detects phosphorylated serine 167 in ERα. (E) ROS levels were detected in live cells using the RedoxSensor Red CC-1 fluorescent dye at 24 and 48 hours post-transfection with either GFP (Vector) or N-174A in the presence or absence of NAC (5 mM). Data are the means±s.d. of the fold increase in ROS (units) relative to the GFP control cells at each timepoint. ***P<0.001 for the ROS generation in cells overexpressing mutant EndoG-N174A at 48 hours compared with at 24 hours. (F) Phosphorylated ERα (serine 167) and total ERα levels were analyzed by western blotting in cells overexpressing either GFP (Vector) or N-174A in the presence or absence of NAC (5 mM). (G) Cells treated as described in E were tested for ER transcription activity as described in A. Data are the means+s.d. of the fold increase relative to control cells overexpressing GFP (Vector) from three independent experiments performed in triplicate. ***P<0.001 for the elevation in ERE luciferase activity compared with that in cells expressing the GFP control vector. (H) Western blotting analysis of total and phosphorylated AKT was performed in cells overexpressing control GFP (Vector), wild-type EndoG–GFP or mutant EndoG-N174A–GFP treated for 24 hours with or without NAC (5 mM). The numbers in D represent the fold induction of the phosphorylated levels of ERα normalized to total levels of ERα in cells transfected with either WT-EndoG or EndoG-N174A relative to control cells transfected with the vector (GFP). The numbers in F represent the fold induction of the phosphorylated levels of ERα normalized to total levels of ERα in cells transfected with EndoG-N174A in the presence or absence of NAC relative to control cells transfected with the vector (GFP). The numbers in H represent the fold induction of the phosphorylated levels of ERα normalized to total levels of ERα in cells transfected with either EndoG-N174A-ΔMLS or EndoG-N174A relative to control cells transfected with the vector (GFP). (I) ER transcription activity was measured by ERE-driven luciferase assay in cells overexpressing the indicated plasmids in the presence or absence of LY (10 μM). Data are the mean+s.d. of the fold increase relative to the control cells overexpressing GFP from three independent experiments performed in triplicate. ***P<0.001 for the elevation in ERE luciferase activity compared with that in cells expressing the GFP control vector. (J) Lysates from cells overexpressing either GFP (Vector), EndoG-N174A or EndoG-N174A that lacks the mitochondrial localization signal (N174A-ΔMLS) were analyzed by western blotting to detect levels of phosphorylated ERα, total ERα, phosphorylated AKT and total AKT using antibodies against phosphorylated ERα (serine 167), ERα, phosphorylated AKT (serine 473) and AKT, respectively.

IMS stress triggers ER-dependent up-regulation of OMI and NRF1. (A) Cell viability was measured in cells at 24, 48, 72 and 96 hours after transfection with either control GFP (Vector) or mutant EndoG-N174–GFP (N-174A) using an MTT assay as described in the Materials and Methods section. Results are the means±s.d. of the percentage cell viability relative to control GFP overexpressing cells. (B) Mitochondrial potential was evaluated in cells transfected with the indicated plasmids followed by TMRE staining and FACS analysis. The percentage of cells without TMRE staining is shown on each panel. (C) Cells transfected with control scramble siRNA (Ctr) or siRNA against ERα, followed by transfection with either control GFP (Vector) or N-174A were used to determine the levels of endogenous OMI by qRT-PCR. ***P<0.001 for the elevation in OMI mRNA compared with that in cells expressing the GFP control vector. (D) Levels of endogenous NRF1 were assessed by qRT-PCR in cells treated as described in C. Data in C and D are the means+s.d. of the fold increase relative to control cells overexpressing GFP from three independent experiments. **P<0.01 for the elevation in NRF1 mRNA compared with that in cells expressing the GFP control vector. (E) Crude extracts from cells treated as described in C were analyzed by western blotting for levels of NRF1, OMI and ERα. The numbers indicate the quantification of the NRF1:tubulin ratio and OMI:tubulin ratio relative to the baseline level set at 1 in control cells overexpressing GFP. The western blot shown is representative of more than three independent experiments. (F) Actin, PHB1 and PHB2 mRNA levels were measured by qRT-PCR. Data represent the means+s.d. of the fold increase relative to control cells overexpressing GFP from two independent experiments.

ERα is required to accelerate proteasome activity and maintain the mitochondrial potential upon IMS stress. (A) Proteosomal chymotrypsin, trypsin and caspase-like activities were assayed in cells overexpressing control GFP (Vector), wild-type EndoG–GFP (WT-Endo G) and mutant EndoG-N174A–GFP (N-174A) as described in the Materials and Methods section. ***P<0.001; *P<0.05 for the elevation in proteasome activity compared with that in cells expressing the GFP vector control. (B) Cells transfected with either scrambled siRNA (Ctr) or siRNA against ERα followed by transfection with the indicated plasmid were used to measure the trypsin-like activity of the proteasome as described in A. ***P<0.001 for the elevation in the trypsin-like activity of the proteasome compared with that in cells expressing the GFP vector control. (C) Viability of cells treated as described in B and exposed to either DMSO or the proteasome inhibitor LLnL was evaluated by the MTT assay. Data in C are the means+s.d. of the percentage cell viability relative to control cells overexpressing GFP from three independent experiments. **P<0.01 reduction in cell viability compared to cells expressing the GFP vector control. (D) Cells lacking ERα and transfected with the indicated plasmids or treated for 24 hours with FCCP (10 μM) were incubated with TMRE and analyzed by FACS to evaluate the mitochondrial potential. The percentage of cells without TMRE staining is shown on each panel. (E) Model of the IMS-stress-initiated signaling cascade, see text for details.