Fig 2: Overexpression of ETV4 partially reverses the inhibitory effect of SLC12A5 knockdown on ferroptosis resistance of BC cells. (A) TBARS assay and (B) C11 BODIPY 581/591 probe were used to assess lipid peroxidation. (C) Levels of Fe2+ in MCF-7 cells transfected with sh-SLC12A5 with or without oe-ETV4. Magnification, ×100. (D) Western blotting was used to evaluate protein levels of SLC7A11, GPX4, ACSL4 and TFR1. *P<0.05, **P<0.01 and ***P<0.001. TBARS, thiobarbituric acid reactive substances; sh, short hairpin; NC, negative control; oe, overexpression; ETV4, E-26-specific sequence variant 4; SLC12A5, solute carrier family 12 member 5; GPX4, glutathione peroxidase 4; ACSL4, Acyl-CoA synthetase long-chain family 4; TFR1, transferrin receptor 1.

Fig 3: Overexpression of ETV4 partially reverses the inhibitory effect of SLC12A5 knockdown on the migration and invasion of breast cancer cells. Cell proliferation was identified by (A) Cell Counting Kit-8 (***P<0.001 vs. Control; ##P<0.01 compared with sh-SLC12A5 + oe-NC) and (B) colony formation assay. (C) Wound healing assay was performed to assess cell migration. (D) Transwell assay was used to detect cell invasion. Magnification, ×100. ***P<0.001 compared with Control or sh-SLC12A5 + oe-NC. sh, short hairpin; NC, negative control; oe, overexpression; ETV4, E-twenty-six-specific sequence variant 4; SLC12A5, Solute carrier family 12 member 5.

Fig 4: Overexpression of ETV4 partially reverses the inhibitory effect of SLC12A5 silencing on glucose metabolism of BC cells. (A) ECAR, (B) OCR, (C) lactic acid production and (D) glucose consumption in MCF-7 cells transfected with sh-SLC12A5 with or without oe-ETV4 were assessed. *P<0.05 and ***P<0.001. ECAR, extracellular acidification rate; OCR, oxygen consumption rate; sh, short hairpin; NC, negative control; ETV4, E-twenty-six-specific sequence variant 4; SLC12A5, Solute carrier family 12 member 5; 2-DG, 2-deoxyglucose; oligo, oligonucleotide.

Fig 5: ETV4 knockdown increases apoptosis and reduces invasion. (A) Annexin V-FITC/PI staining showed that shETV4 increases cell apoptosis in glioma cells. (B) Quantification of apoptosis for panel (A). (C) Transwell invasion assays showed that shETV4 reduced cell invasion in glioma cells. (D) Quantification of invasion for panel (C) *P < 0.05; **P < 0.01.