Fig 2: Cisplatin-resistant CRC cells exhibit elevated glutamine metabolism(A) Heatmap of differentially expressed genes from LoVo parental and CDDP R cells identified by RNA-seq. (B) MetaboAnalyst pathway enrichment analysis of metabolites in LoVo parental and CDDP R cells. (C) mRNA expression levels of glutamine metabolism enzymes and regulators were detected by qRT-PCR in LoVo parental and CDDP R cells. (D) Western blot results show protein expression of GLS1 and GLS2 in LoVo parental and CDDP R cells. (E and F) Glutamine uptake (E) and GLS activity (F) assays were performed in LoVo parental and CDDP R cells. (G) LoVo parental and CDDP R cells were cultured with regular medium or glutamine depletion medium, a clonogenic assay and (H) annexin V assay were performed. (I) LoVo CDDP R cells cultured with regular medium or glutamine depletion medium were treated with cisplatin at 10 and 20 μM for 48 h. (J) Cell viability and cell death were examined by a clonogenic assay and (H) annexin V assay. Columns include mean of three independent experiments; data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 3: Change of protein levels of PER3, GLS2, CREM and TXNIP in AT1 prostate and Walker-256 mammary carcinoma cells after 24 h under acidotic (pH 6.6) and/or hypoxic (pO2 = 1.5 mmHg) conditions. n = 4–12; (*) p < 0.05, (**) p < 0.01 vs. control; (#) p < 0.05, (##) p < 0.01 AT1 vs. Walker-256 cells

Fig 4: Protein levels of PER3, GLS2, CREM and TXNIP in AT1 prostate and Walker-256 mammary carcinoma tumors in vivo 24 h after (a) inducing metabolic acidosis by forcing glycolytic metabolism or (b) intratumoral injection of lactic acid. n = 4–18; (*) p < 0.05, (**) p < 0.01 vs. control; (#) p < 0.05, (##) p < 0.01 AT1 vs. Walker-256 cells

Fig 5: YTHDF1-mediated cisplatin resistance is through promoting glutamine metabolism(A) DLD-1 cells were transfected with control or YTHDF1 overexpression plasmid for 48 h. Cells were treated without or with BPTES for 24 h. The GLS activity was measured. (B) The above cells were treated with cisplatin at 0, 2.5, 5, 10, 20, or 40 μM for 48 h. Cell viability and death were assessed by an MTT assay and (C) annexin V assay, respectively. (D) LoVo cells were transfected with control or YTHDF1 overexpression plasmid for 48 h. Cells were treated without or with BPTES for 24 h. The GLS activity was measured. (E) The above cells were treated with cisplatin at 0, 2, 4, 8, 16, or 32 μM for 48 h. Cell viability and death were assessed by an MTT assay and (F) annexin V assay, respectively. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.