Fig 1: A) Immunofluorescence staining of three control fibroblast lines, as well as the ATP7AM1311V cells. All fibroblasts were treated with either 0.5 μM CuCl2 (basal copper) or 200 μM for 2 h, then fixed and stained with antibodies against ATP7A, the Golgi marker TGN46 as well as DAPI and imaged on a confocal microscope. Shown are representative images from one control line and the ATP7AM1311V. The scale bars denote 20 μm and 5 μm for the enlarged images. B) Pearson’s correlation coefficient (PCC) of colocalization was computed for the various fibroblast lines under basal copper conditions, comparing the average of the three control fibroblast lines and the ATP7AM1311V fibroblasts. 20–35 cells were measured per line (control is the average of three control lines). C) PCC of colocalization was used to measure colocalization of ATP7A and TGN46 under basal and 200 μM copper conditions. Asterisks denote significance within each cell line, comparing the treated and untreated conditions (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). 50–100 cells were measured per line per condition.
Fig 2: Western blot performed on total lysate(s) from wild-type and edited HepG2 cells.Immunoblot for ATP7B shows decreased protein expression in 2F3, 1F6 and 1E8 cell lines compared to HepG2 wild-type cells; protein expression in 1F6 and 1E8 homozygous lines is higher than in 2F3 compound heterozygous, as expected. No observed expression in the 2A1 knock-out (k/o) cell line. Immunoblot for beta-actin was used as a protein loading control.
Fig 3: Elesclomol‐induced loss of ATP7A is required for SLC7A11 degradation. (A) Exogenous ATP7A was overexpressed in cells treated with 20 nm elesclomol for 24 h followed by Western blot to show the protein expression of SLC7A11 (n = 3). (B) Endogenous ATP7A was knocked down by siRNA and then treated as (A) (n = 3). (C) Endogenous ATP7A was knocked down in indicated cells followed by co‐IP assay to determine the ubiquitination of SLC7A11 (n = 3). (D, E) Levels of Cu2+ in cells and medium were measured after the knockdown of ATP7A (n = 3). (F) Immunofluorescence assay showing the ROS level in cells with or without knockdown of ATP7A (n = 3). Scale bar, 50 μm. (G) Cells in (C) were treated with or without antioxidant NAC (1 mm) for 24 h followed by detection of the ubiquitination of SLC7A11 (n = 3). (H) Exogenous ATP7A was overexpressed in cells treated with 20 nm elesclomol for 24 h followed by co‐IP assay to determine the ubiquitination of SLC7A11 (n = 3). (I) Immunofluorescence assay showing lipid ROS level in cells with or without overexpression of exogenous ATP7A (n = 3). PE (red) and FITC (green) represent reduced and oxidized form of probes, respectively. Scale bar, 50 μm. Data are means ± SEM from at least three independent repeats. The P‐values were determined by two‐tailed t‐test. **P < 0.01; ***P < 0.001.
Fig 4: Elesclomol inhibits CRC through degradation of ATP7A. (A) Survival fraction assay showing the cell survival on treatment with elesclomol (20 nm) and Cu2+ (2 μm) for 24 h with or without the overexpression of exogenous ATP7A (n = 3). (B) Cells were treated as (A) with or without the knockdown of endogenous ATP7A (n = 3). (C, D) MTT assay showing the cell viability of cells treated as (A) and (B) (n = 3). (E) Cells were treated with indicated copper chelators (20 nm) for 24 h with or without the supplementation of 2 μm Cu2+ followed by immunoblotting to determine the expression of ATP7A (n = 3). (F) Survival fraction assay showing the cell survival in the treatment of indicated copper chelators (20 nm) in combination with 2 μm Cu2+ or ATP7A silencing (n = 3). (G) Working model of elesclomol‐induced ATP7A degradation and CRC ferroptosis. Elesclomol elevates the Cu2+ level in mitochondria and decreases the expression of ATP7A, leading to Cu2+ retention within cells and consequent ROS accumulation. This effect promotes the degradation of SLC7A11, which further enhances oxidative stress, eventually leading to ferroptosis in CRC cells. Data are means ± SEM from at least three independent repeats. The P‐values were determined by two‐tailed t‐test. ***P < 0.001.
Fig 5: A) Control and two different clones of ATP7AM1311V iPSCs (A and B) were differentiated into motor neurons, and cells were lysed for total protein extraction at days 58–66 of differentiation. Two independent differentiations were performed and western blot for the various indicated proteins were performed. B) RNA was prepared from two independent differentiations of control and ATP7AM1311V iPSC-MN as in A) and mRNA levels of the indicated genes were measured, normalized to GAPDH control, and plotted as fold increase of M1311V iPSC-MNs over control MNs. C) Control and ATP7AM1311V iPSC-MN monocultures were prepared and localization of ATP7A, TGN46, and DAPI was observed by immunohistochemistry at day 60 of differentiation. Neurons were identified by MAP2 positive staining (not shown). Shown are representative images from control and one ATP7AM1311V iPSC-MN clone, post-treatment with basal copper (0.5 μM) for 3 h, 200 μM CuCl2 for 3 h, or the latter, followed by copper washout for 3 h. Scale bars denote 15 μm. D) PCC of colocalization of ATP7A and TGN46 at basal copper conditions was calculated for 50–80 cells from 1 control line and 2 clones of the same ATP7AM1311V iPSC-MN line, cultured on mouse astrocytes. Asterisks denote significance (*p < 0.05, **p < 0.01, *** = p < 0.001, **** = p < 0.0001). E) PCC of colocalization was computed similar to D) for iPSC-MNs cultured without astrocytes, under basal copper conditions (0.5 μM), treated with 200 μM CuCl2 for 3 h, or treated with 200 μM CuCl2 for 3 h followed by copper washout for 3 h. 30–35 cells from the same control line and two ATP7AM1311V clones at day 57 of differentiation were measured. Asterisks denote significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
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