Journal: The FASEB Journal
Article Title: Elesclomol‐Induced Copper Influx Attenuates Lung Adenocarcinoma Progression With Involvement of the ER Stress/PCK2 Axis
doi: 10.1096/fj.202502105RR
Figure Lengend Snippet: ES induces copper‐specific death in LUAD cells. (A) CCK‐8 assays revealed that ES (100 nM) inhibited only cell viability (to 20%) when combined with copper ions, whereas it failed to inhibit cell viability when combined with other metal ions (Fe 3+ , Co 2+ , Ni 2+ , and Zn 2+ , 1 μM). (B) CCK‐8 assay results indicating that pulse treatment with ES + Cu (100 nM, 1:1)for 2 h, followed by washing with PBS and replacing with fresh medium, led to persistent inhibition of cell viability at 8, 24, and 48 h. (C, D) Flow cytometry analysis indicated that treatment with ES + Cu (100 nM, 1:1)did not significantly increase the Annexin V + /FITC + proportion of early apoptotic cells. (E) Western blot analysis revealed that treatment with ES + Cu (100 nM, 1:1) did not increase the expression level of the apoptosis marker cleaved PARP, suggesting that the cell death mediated by ES + Cu was not via the classic apoptotic pathway. (F) The cell death induced by ES + Cu (100 nM, 1:1) could not be reversed by other cell death inhibitors but could be reversed by the copper chelator TTM, indicating that the cell death mediated by ES + Cu was copper ion dependent and not via other programmed cell death pathways. Before ES + Cu (100 nM, 1:1) treatment, the cells were pretreated overnight with 20 μM necrostatin‐1 (Nec‐1), 10 μM ferrostatin‐1 (Fer‐1), 1 mM N‐acetylcysteine (NAC), 30 μM Z‐VAD‐FMK, 50 μM D‐Boc‐FMK, 20 μM TTM, 300 μM L‐NAME, or 1 μM pepstatin A. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was determined using unpaired two‐tailed Student's t ‐test. ns: p > 0.05, *** p < 0.001.
Article Snippet: Elesclomol (#STA‐4783; Selleck Chemicals, Houston, TX, USA), tetrathiomolybdate (#E1166; Selleck Chemicals, Houston, TX, USA), acetylcysteine (N‐acetylcysteine) (#S1623; Selleck Chemicals, Houston, TX, USA), necrostatin‐1 (#S8037; Selleck Chemicals, Houston, TX, USA), pepstatin A (#S7381; Selleck Chemicals, Houston, TX, USA), D‐Boc‐FMK (#HY‐13229; MedChemExpress, NJ, USA), Fer‐1 (ferrostatin‐1) (#S7243; Selleck Chemicals, Houston, TX, USA), Z‐VAD‐FMK (#S7023; Selleck Chemicals, Houston, TX, USA), L‐NAME HCl (#S2877; Selleck Chemicals, Houston, TX, USA), 4‐phenylbutyric acid (#HY‐A0281; MedChemExpress, NJ, USA), thapsigargin (#HY‐13433; MedChemExpress, NJ, USA), CuCl 2 (CAS: 7447‐39‐4; Aladdin, Shanghai, China), CoCl 2 (CAS: 7646‐79‐9; Aladdin, Shanghai, China), FeCl 3 (CAS: 7705‐08‐0; Aladdin, Shanghai, China), NiCl 2 (CAS: 7718‐54‐9; Aladdin, Shanghai, China), and ZnCl 2 (CAS: 7646‐85‐7; Aladdin, Shanghai, China)were used under experimental conditions in vitro and in vivo.
Techniques: CCK-8 Assay, Inhibition, Flow Cytometry, Western Blot, Expressing, Marker, Two Tailed Test
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