Fig. 1: The nuclear localization of PYCR1 is critical for tumor cell growth under hypoxia.

b–g immunoblotting and immunoprecipitation analysis were performed using the indicated antibodies. a, h, i the values are presented as mean ± s.d.; statistical analysis was performed using the two-tailed Student’s t test or Pearson’s chi-squared test. Source data are provided as Source Data uncropped western blots and Source Data Fig. 1. a IHC analysis was performed in 150 human colorectal tumor specimens, representative images are shown (upper panel, n = 150; middle panel, for I+II, n = 81, in which low and high, respectively, are 45 and 36, for III+IV, n = 69, in which low and high, respectively, are 13 and 56; bottom panel, for Low, n = 58, for High, n = 92). Scale bars, 100 μm. b HCT116 cells were cultured under normoxia or hypoxia for 12 h and cytosolic and nuclear extracts were collected. c HCT116 cells expressing indicated Flag-tagged PYCR1 were cultured under hypoxia for 12 h, and nuclear extracts were collected. d HCT116 cells expressing Flag-tagged PYCR1 were cultured under normoxia or hypoxia for 12 h. Nuclear extracts subjected to immunoprecipitation with the anti-Flag antibody were analyzed by silver staining. e HCT116 cells were cultured under normoxia or hypoxia for 12 h. Immunoprecipitation analysis was performed post collection of cytosolic (left panel) or nuclear extracts (right panel). f HCT116 cells transfected with IGF1R shRNA (left panel) or PYCR1 shRNA (right panel) were cultured under normoxia or hypoxia for 12 h. g HCT116 cells were pretreated with PPP (1 μM), ARQ-092 (2 μM) and GDC-0994 (10 μM) for 1 h before being cultured under hypoxia for 12 h. h WT and mutant His-PYCR1 were purified and the enzymatic activity was measured (n = 3 independent experiments). i HCT116 cells with depletion of PYCR1 and reconstituted with an expression of indicated Flag-rPYCR1 were treated with or without exogenous proline (3 mM). Cells were cultured under normoxia or hypoxia for 48 h. Cellular viability was examined by CCK-8 assay (n = 5 independent experiments).