Fig 2: NRF1 degradation is required for tumor maintenance and TAM polarization. a–c Images (a), growth curves (b) and weights (c) of xenograft tumors derived from MDA-MB-231 cells with the indicated modifications. Tumors were established in mice by subcutaneous injection of cells. d, e The indicated xenograft tumor tissues were analyzed by hematoxylin-eosin staining (d) and tissue cross-sections were quantified necrotic area (e). Scale bars, 500 µm. f The indicated xenograft tumor tissues were stained with anti-ARG1 (red) and anti-TOMM20 (green) antibodies together with DAPI (blue). Scale bars, 50 µm. g <3 KDa fractions from DMEM or indicated cell-conditioned medium were used to stimulate bone-marrow derived macrophages. ARG1 mRNA expression was analyzed by qRT-PCR and normalized to ATCB as housekeeping gene. h Tissues derived from mouse spontaneous breast cancer were stained with DAPI (blue) together with anti-ARG1 (red) and anti-TIMM23 (green) or anti-GLUT1 (red) antibodies. Scale bars, 25 µm. For all panels, error bars indicate s.d. For panel (a–f) and (h), n = 5 mice per group. For panel (g), n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used. The two-tailed unpaired student t-test was used to compare data

Fig 3: NRF1 Lys230 is responsible for SIAH2-mediated NRF1 ubiquitination and degradation under hypoxia. a Determination of the Lysine 230 of NRF1 is responsible for SIAH2-induced degradation. Quantification of NRF1 protein levels is shown below. b SIAH2 induces degradation of wild-type NRF1 in a dosage-dependent manner but has no effect on NRF1-K230R. c SIAH2 induces ubiquitination of NRF1 but not NRF1-K230R. d Purified SIAH2 promotes ubiquitination of bacterially expressed wild-type NRF1 but not NRF1-K230R in vitro. e Co-immunoprecipitation of exogenously expressed Myc-NRF1 or Myc-NRF1-K230R with Flag-SIAH2RM. f Hypoxia-induced ubiquitination of NRF1 is abolished by expression of the NRF1-K230R mutant in vivo. g HeLa cells were transiently transfected with wild-type NRF1 or NRF1-K230R for 12 h, and then incubated under normoxia or hypoxia for an additional 36 h. Cells were harvested and analyzed by immunoblotting with the indicated antibodies. Quantification of NRF1 protein levels is shown below. For all panels, error bars indicate s.d., n = 3 biological replicates. Data were compared with two-tailed paired ratio t-tests

Fig 4: SIAH2 promotes NRF1 polyubiquitination and degradation under hypoxia. a Hypoxia-mediated NRF1 degradation is inhibited by the proteasomal inhibitor MG132, but not by the autophagy inhibitor Bafilomycin A1 (BafA1). Right: densitometric quantification of NRF1 expressions. b MDA-MB-231 cells were incubated under normoxia or hypoxia for 24 h. Cells were treated with 10 µM MG132 for 6 h before harvesting. Cell lysates were immunoprecipitated with anti-NRF1 antibodies and then detected by western blotting with anti-NRF1 and anti-Ubiquitin antibodies. c Hypoxia-related ubiquitin E3 ligases were transiently co-transfected with Myc-NRF1 into HeLa cells for 24 h, and Myc-NRF1 protein levels were detected by western blotting with anti-Myc antibodies. d Direct interactions between bacterially expressed His-NRF1 and GST-SIAH2 in vitro. e Ectopic expression of SIAH2, but not SIAH2RM increased NRF1 ubiquitination in vivo. f Ubiquitination of bacterially expressed His-NRF1 by purified SIAH2 but not by SIAH2RM in vitro. g MDA-MB-231 cells were cultured under normoxia or hypoxia for 18 h, then treated with 10 µM MG132 and incubated under normoxia or hypoxia for another 6 h. Endogenous interactions between NRF1 and SIAH2 were analyzed by immunoprecipitation. h Wild-type or SIAH2−/− MDA-MB-231 cells were transiently transfected with scramble or NRF1-targeted siRNA, and then cultured under normoxia or hypoxia for 36 h. Cells were harvested and analyzed by western blotting. Bottom: densitometric quantification of the indicated proteins. i Hypoxia-induced ubiquitination of NRF1 is abolished by depletion of SIAH2 in vivo. j Top: representative immunohistochemical staining of NRF1 and SIAH2 in normal breast tissue and breast cancer tissue from the tissue microarray. (Brown color indicates positive immune reaction; scale bars, 50 µm). Bottom: heat map showing quantitative analysis of the expression of NRF1 and SIAH2 proteins in normal breast tissues and breast cancer tissues. n = 158 breast tumors and n = 27 normal breast samples. Statistical analysis of the immunostaining results is shown in Supplementary Table 3, 4. For all panels, error bars indicate s.d., n = 3 biological replicates. Data were compared with two-tailed paired ratio t-tests

Fig 5: Markers of mitochondrial biogenesis and mitophagy. Markers of mitochondrial biogenesis (a, b) and mitophagy (c, d), with representative western blot images (e). No significant differences were detected for markers of mitochondrial biogenesis or mitophagy in the cortex and cerebellum. Data are presented as mean ± SD and individual data points. YNG, young (n = 10); MA‐SED, middle‐aged sedentary (n = 12); MA‐PA, middle‐aged physical activity (n = 12). Note that the following proteins share the same ponceau: Cortex: PGC‐1a and SOD2; TFAM and OPA1; Parkin, Mfn2, and Fis1; Cerebellum: PGC‐1a and SOD2; NRF1 and PINK1; TFAM and OPA1.