Fig 1: In vitro cell invasion in Saos-2 and MG-63 cells. (A) In Saos-2 cells, in vitro cell invasion assays were performed in normal control cells (NC), cells stably transfected with empty pcDNA3 vector (VC), and cells stably transfected with pcDNA3-AEG-1 expression vector (AEG-1) with or without LY294002 (LY; 50 µM) or BQ123 (BQ; 5 µM) treatment. (B) In MG-63 cells, in vitro cell invasion assays were performed in NC, cells stably transduced with scrambled control shRNA (SC), and cells stably transduced with AEG-1-shRNA (A-shRNA) with or without ET-1 (10 pM and 100 pM) treatment alone or ET-1 (100 pM) combined with LY (50 µM) or BQ (5 µM). Cells treated with LY (50 µM) or BQ (5 µM) alone were also analyzed. Invasion cell numbers were counted and the cell invasion level is shown as the fold change in invasion cell number relative to that of NC (designated as 1). aP<0.05 compared with (A) NC and VC or (B) SC; bP<0.05 compared with (A) AEG-1 or (B) A-shRNA and ET-1 (10 pM) ; cP<0.05 compared with A-shRNA and ET-1 (100 pM); dP<0.05 compared with A-shRNA, ET-1 (100 pM) and LY. ShRNA, short hairpin RNA.
Fig 2: MTDH induces metabolomic changes that increase the susceptibility to ferroptosis.a. Using the MDA-MB-231 cell line, knocking out MTDH increased intracellular cysteine and methionine, but reduced glutamate and a-KG levels. b. MTDH KO in MDA-MB-231 cells resulted in elevated levels of glutathione. c. Similar results were observed in Hec50 cells. *P < 0.05, **P < 0.01 by two-way ANOVA
Fig 3: Mesenchymal-high cancers exhibit enhanced sensitivity to ferroptosis inducers.a Representative cell lines showing enhanced sensitivity to GPx4 inhibitor ML162 correlates to low levels of E-cadherin, an epithelial marker. H1975 and A549: lung cancer; KLE and Ishikawa: endometrial cancer. Antibodies against E-cadherin, ZEB1, vimentin, and MTDH were used to detect protein extracts from non-small-cell lung cancer H1975 and A549, and endometrial cancer cell lines KLE and Ishikawa. ß-Actin was detected as a loading control. b Summary of ML162 sensitivity in correlation to E-cadherin expression (R: resistant, n = 4; S: sensitive, n = 7; ****P < 0.0001 by two-sided paired t test). Refer to Supplementary Table 1 for details. c Z-score-transformed Pearson’s correlations between compound AUCs and the mesenchymal score from single-sample GSEA across 481 different compounds and 516 cell lines derived from CTRP. The GPx4 inhibitors ML162, ML210, and RSL3 are highlighted in red. d Correlation of CDH1 expression vs. AUC for erastin, a system xc- inhibitor, across carcinoma cell lines. This is visualizing the type of analysis performed in C, but at an individual molecule level and using CDH1 as a proxy for EMT
Fig 4: MTDH confers enhanced sensitivity to ferroptosis inducers in vitro.a, b ML162. c, d ML210. a, c MDA-MB-231. b, d Using Hec50. In all cases, as compared to MTDH KO cells, MTDH-high WT cells exhibited enhanced sensitivity to GPx4 inhibitors. WT: wild type; KO: CRISPR knockout isogenic cells
Fig 5: MTDH levels correlate with the ferroptotic effect.a Using KLE cells, MTDH levels were reduced with increasing doses of ML162 and ML210, which could be reserved by ferroptosis inhibitor ferrostatin-1 (1 µM). b Similar changes were observed when ML162 was used in combination with other ferroptosis inducers (sorafenib: 5 µM; erastin: 2.5 µM; SAS: 400 µM) in MDA-MB-231 and KLE cells. c MTDH levels were reserved by ferrostatin-1 (1 µM) with a combination treatment. d However, such correlation between MTDH levels and dose of ferroptosis inducers were not observed in ferroptosis-resistant Ishikawa cells
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