Fig. 3: ATP7A protects KRAS-mutant cells from cuproptosis.

a Micrographs showing ATP7A expression (green) and DAPI (blue) in Control and KRAS cells. Scale = 20 µm, n = 3 independent experiments. b Cellular distribution profile of ATP7A as seen in a for Control (blue line) versus KRAS (red line) cells. Data were normalized to KRAS. PM plasma membrane. c IB for CCS and ATP7A expression in Control and KRAS cells, with CCS quantifications (right). All panels represent n = 7 independent experiments. *P = 0.0110. d Time-course of average crisp-17 fluorescence ratios (n = 10 cells) upon addition of DTDP at T = 0 min. Yellow arrows indicate timepoints for which quantifications are shown (right). P-values are represented as Control-basal versus DTDP (6 min) (&), Control-DTDP (6 min) versus KRAS-DTDP (6 min) (#), KRAS-basal versus DTDP (6 min) ($), Control-DTDP (6 min) versus DTDP (20 min) (^), KRAS-DTDP (6 min) versus DTDP (20 min) (%). e Graph depicting relative cell viability after CuCl2 treatment. Data represent n = 6 independent experiments.***P = 0.0002 (Control versus KRAS-5); ***P = 0.0006 (Control versus KRAS-10). f IB depicting ATP7A and CCS levels in the indicated CRC cells. Data represent n = 3 independent experiments. g As in d but for CRC (n = 14 cells). P-values are represented as CACO-2-basal versus DTDP (4 min) (ns), CACO-2-DTDP (4 min) versus HCT116-DTDP (4 min) (#), HCT116-basal versus DTDP (4 min) ($), CACO-2-DTDP (4 min) versus DLD-1-DTDP (4 min) (&), DLD-1-basal versus DTDP (4 min) (+), CACO-2-DTDP (4 min) versus DTDP (30 min) (ns), HCT116-DTDP (4 min) versus DTDP (30 min) (^), DLD-1-DTDP (4 min) versus DTDP (30 min) (%). h As in e but for CRC cells. Data represent n = 3 independent experiments. i, j Graphs represent expression of cell-surface ATP7A (i, n = 40 random images from five patient-derived CRC tumors) and total CCS (j, n = 35) analyzed by immunohistochemistry. For c–e, g–j center values and error bars represent mean ± SEM and for left panels (d, g), mean ± SD. For d, g, h ns not-significant or ****P < 0.0001 (&, #, +, $, ^, %). Significance was determined using unpaired two-tailed Student’s t-tests (c, d, g, i, j) or two-way ANOVA with post-hoc Bonferroni’s multiple-comparison analysis (e, h).