Fig 1: LOX inhibition generates vulnerability to targeting DHODH-mediated ferroptosis defense in TNBCs(A) WB of KEAP1, FSP1, and GPX4 in MDA-MB-231 cells upon LXG6403.(B) Intracellular GSH/GSSG ratio upon LXG6403 (n = 3 technical replicates).(C) WB of FSP1, GPX4, and DHODH in MDA-MB-231 cells upon LXG6403 (5 and 10 μM) with or without mdivi-1 (10 μM).(D) WB of GPX4 in cytosolic/mitochondrial fractions upon LXG6403.(E) WB of GPX4 and FSP1 in shLOX cells expressing WT or S65A PARKIN.(F) IF images of FSP1 and TOM20 upon LXG6403 treatment with or without MG132. Scale bars: 25 μm.(G) Cytosolic ROS in shLOX expressing MDA-MB-231 cells transfected with WT or S65A mutant PARKIN (n = 2 technical replicates).(H) Network of metabolites upregulated upon LXG6403 (15 μM) for 24 h in MDA-MB-231 cells (n = 3 biological replicates).(I) Relative DHODH activity in BT549 cells treated with LXG6403 (15 μM for 24 h), mdivi-1 (10 μM), or both (n = 3 technical replicates).(J and K) Relative mitochondrial ROS (J) and lipid peroxidation (K) in MDA-MB-231 cells treated with the combination of LXG6403 (15 μM, 24 h) and brequinar (50 μM, 24 h) (n = 3 technical replicates).(L) Percentage viability inhibition upon treatment with multiple doses of brequinar and overexpression of HIF1αΔODD, S65A PARKIN, or LOX/HSP90 in shLOX cells (n = 2 biological and 8 technical replicates).(M) Relative αKG levels upon tetracycline-inducible LOX knockdown followed by overexpressing HIF1αΔODD, S65A PARKIN, or LOX/HSP90 (n = 2 biological and 6 technical replicates).(N–P) Percentage ATP production inhibition (N) (n = 3 technical replicates), cytosolic ROS (O) (n = 2 technical replicates), and WB of GPX4 (P) upon tetracycline inducible LOX knockdown in MDA-MB-231 cells.(Q) Percentage viability inhibition following treatment with multiple doses of brequinar in combination with LXG6403 with or without αKG supplementation (n = 4–6 technical replicates).(R) Percentage viability inhibition in BT549 cells treated with multiple doses of brequinar in combination with LXG6403 with or without ferroptosis inhibitors ferrostatin-1 (Fer-1, 100 nM) or liproxstatin-1 (Lip-1, 200 nM) for 3 days (n = 3–6 technical replicates). Data are presented as mean values ± SD. p values were calculated with unpaired, two-tailed Student’s t test (B, G, I, J, K, L, M, N, O, Q, and R). ∗p < 0.05; ∗∗p < 0.01.
Fig 2: LOX promotes MERCs by preventing PARKIN-mediated VDAC1 degradation and increases mitochondrial Ca2+ transport by interacting with HSP90(A) Experimental pipeline of LOX-BioID in MDA-MB-468 cells (n = 3 biological replicates).(B) Network analysis of the LOX interactome identified by BioID followed by mass spectrometry.(C) Pathway enrichment analysis of LOX-interactome.(D) PLA of VDAC1 and IP3R in MDA-MB-468 cells overexpressing LOX and/or HSP90 upon LXG6403. Scale bars: 25 μm.(E) Quantification of PLA in (D) (n = 6 images).(F) VDAC1 ubiquitination in cells expressing WT or S65A PARKIN upon tetracycline and MG132.(G) WB of VDAC1 in WT and S65A PARKIN-expressing cells upon LXG6403.(H) WB of VDAC1 in Ctrl or VDAC1 ORF-transfected Hs578T cells treated with LXG6403.(I) Quantification of MERC formation in Ctrl or VDAC1 ORF-transfected Hs578T cells treated with LXG6403 (n = 3 technical replicates).(J) PLA of LOX and HSP90 in LOX-overexpressing MDA-MB-468 cells. Scale bars: 25 μm.(K) Quantification of PLA in (H) (n = 8 images).(L and M) Mitochondrial (L) and cytosolic (M) Ca2+ levels upon tetracycline inducible LOX knockdown with or without LOX reconstitution or 17-AAG. (n = 3 technical replicates).(N) Reciprocal co-IP showing the interaction between HSP90 and IP3R in BT549 cells upon LXG6403.(O) Relative αKG levels in cells from (L) (n = 3 technical replicates).(P) % inhibition of ATP production in cells from (L) (n = 4 technical replicates). Data are presented as mean values ± SD. p values were calculated with unpaired, two-tailed Student’s t test (E, I, K, L, M, O, and P). ∗p < 0.05; ∗∗p < 0.01.
Supplier Page from Abcam for Recombinant Human HSP90 alpha Protein (His-tag)