Extended Data Fig. 2: Hypoxia promotes the expression of CARNS2, which can facilitate tumor progression.
From: Carnosine regulation of intracellular pH homeostasis promotes lysosome-dependent tumor immunoevasion
a, Immunoblotting analysis of different isoforms of CARNS in mouse tissues (left). Western blot analysis of different isoforms of CARNS in human brain, liver and breast tissues. Adjacent non-cancerous tissues (N), tumor tissues (T) (right). b, Immunoblotting analysis of CARNS in HepG2 cells expressing shCARNS1 or sgCARNS1. c, qPCR analysis of CARNS expression in HepG2 and PLC cells. Cells were treated under normoxia or hypoxia for 48 h (n = 3 biological replicates). d, ChIP experiments were performed in HepG2 and PLC cells expressing Flag-HIF-2a or EV using anti-Flag antibody. The occupancy of potential binding sites in CARNS by HIF-2a was determined by qRT-PCR (n = 3 biological replicates). e, Dual-luciferase analysis in HEK293 cells transfected with pGL3-HRE and HIF-1α or HIF-2α plasmids (n = 4 biological replicates). f, View of aligned reads at 20 kb resolution for a matched hypoxia/normoxia pair of CARNS. Paired arrows represent semi-RT-PCR primers. g, Schematic diagram of isoforms of CARNS. h, i, Intracellular carnosine levels were detected in HepG2 cells stably expressing EV, CARNS1-V1, CARNS1-V2 and CARNS2 (n = 3 biological replicates) (h). Immunoblotting analysis confirmed overexpression of CARNS1-V1, CARNS1-V2 and CARNS2 in HepG2 cells (i). j, Plasmids expressing RFP or YAP-5SA together with PB transposase plasmids were delivered into WT, Carns1−/− or Carns1/2−/− mice by hydrodynamic injection. Liver tumors were analyzed at 100 days after injection. Red fluorescent protein (RFP) served as a control (n = 5 mice). Data presented as mean ± s.d. (c, d, e, h). Statistical significance was determined by two-tailed unpaired Student’s t-test (c, d, e, h).
