Fig 1: SLC38A9 and RagC as crucial genes for NRF3-mediated arginine-dependent mTORC1 activation(A) The effect of amino acid or arginine depletion on the nuclear localization of NRF3. Related to Figure S2F, H1299-oeNRF3 cells were transfected with siCtrl for two days. For amino acid (AA) or arginine (Arg) depletion, the cells were cultured without all amino acids or arginine for 24 h (AA or Arg depletion). As a positive control, the cells were incubated with 1 µM MG-132 for 24 h. Scale bars, 10 µm.(B) The effect of NRF3 knockdown on the expression of five Ragulator-associated genes in response to arginine stimulation. For two days, HCT116 cells were transfected with the indicated siRNA. The cells were cultured without arginine for 5 h and then restimulated with arginine for 15 min.(C) The binding potential of NRF3 onto the SLC38A9 or RagC promoter. H1299-oeNRF3 or oeGFP cells were treated with 1 µM MG-132 for 16 h. The ChIP-regions of each gene are shown in Figure S3B.(D) The effect of NRF3 knockdown on the expression of SLC36A1 gene in response to amino acid (upper graph) or arginine (lower graph) stimulation. HCT116 cells were transfected and stimulated as in (B).(E) The binding potential of NRF3 onto the SLC36A1 promoter. H1299-oeNRF3 or oeGFP cells were treated as in (C). The ChIP region is shown in Figure S3B.(F) The effects of the indicated gene knockdown on mTORC1 activation in response to amino acid stimulation. HCT116 cells were transfected and stimulated as in (B).(G) The add-back effect of SLC38A9 and RagC genes on arginine-dependent mTORC1 activation in HCT116-siNRF3 cells. HCT116 cells were transfected with p3×FLAG-CMV 10 containing full-length (FLAG-GFP), pRK5 FLAG-SLC38A9.1 (FLAG-38A9, black arrowhead), and/or pRK5 FLAG-RagC (FLAG-RagC, white arrowhead). One day after plasmid transfection, the cells were transfected with the indicated siRNA for two days and then stimulated with arginine, as shown in (B).(C and E) Welch t-test, (B and D) ANOVA followed by Tukey’s test: ***p< 0.005; *p< 0.05; n.s., not significant (n = 3, Mean ± SD). See also Figures S2 and S3.
Fig 2: NRF3-mTORC1 axis aberration causes tumor growth and poor prognosis(A) The effect of indicated gene knockdown or mTORC1 inhibition on the in vitro growth of cancer cells under amino acid stimulation. HCT116 cells were transfected with the indicated siRNA for two days. Next, the cells were cultured without all amino acids for 5 h, then restimulated with all amino acids for 15 min. Subsequently, the cell numbers were counted using a hemocytometer. As a control, HCT116-siCtrl cells were treated with 10 µM rapamycin for two days and amino acid stimulation (n = 3, Mean ± SD).(B and C) The effect of mTORC1 inhibition on NRF3-dependent tumor growth in mouse xenograft models. H1299-oeGFP and H1299-oeNRF3 cells were injected subcutaneously into BALB/cAJcl-Foxn1nu mice, and then, rapamycin (1.5 mg/kg or 3.0 mg/kg) was intraperitoneally administered once every two days. Photographs and weights of tumors four weeks after injection are shown in (B) and (C). In (B), Scale bars, 5 mm. (n = 5–6, Mean ± SD).(D) Overall or disease-free survival significance maps of the eight indicated genes in various cancer types, estimated using the Mantel–Cox test with a Hazard ratio (HR).(E) Kaplan–Meier plots comparing overall or disease-free survival of mesothelioma (MESO) patients with lower or higher expression levels of eight signatures, involving NRF3, RagC, SLC36A1, SLC38A9, RAB5A RAB5B, RAB5C, and SLC7A1 genes.(F) A schematic showing NRF3-mediated arginine-dependent mTORC1 activation for cancer development. See the discussion section for details. (A, C) ANOVA followed by Tukey’s test: ***p< 0.005; n.s., not significant. See also Figure S7.
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