Fig. 5.

Characterization of Cys regulation of bystander uptake. a Cysteine presence is necessary for the increase of Ag-647 bystander uptake. CHO (left) and H1975 (right) cells were pre-incubated in Cys medium before exposed to Ag-647 and T-Ag in AA-free (Cys to AA-) or Cys medium (Cys to Cys). b The bystander uptake of Ag-647 was increased as Cys concentration increased. CHO cells were incubated with Ag-647 and T-Ag in AA-free medium with gradually increasing concentrations of Cys from 0 to 2 mM. c T-Ag-induced bystander uptake is stimulated by cystine and GSH. CHO (left) and H1975 (right) cells were incubated in cystine or GSH containing medium with Ag-647 and T-Ag. d Bystander uptake of Ag-647 was inhibited by cystine inhibitors. CHO cells were pre-treated with erastin or glutamate for 10 min prior to the incubation with Ag-647 and T-Ag for 30 min in the presence of erastin or glutamate. Left, bystander uptake of Ag-647; Right, uptake of corresponding T-Ag. For all above panels, the cells were etched after incubation, and the fluorescence intensity per cell of internalized NPs was determined by flow cytometry, and normalized to that of Ag-647 (For Ag-647 bystander uptake in a–d (left)) or Ag-555 (For T-Ag uptake in d(right)) alone. All quantified data a–d were analyzed using one-way ANOVA with Tukey’s multiple comparisons and are presented as mean ± s.d. (n = 6 for a, c, and d; n = 5 for b). One-way ANOVA, a (left), F = 40.268, P < 0.0001; a (right), F = 160.20, P < 0.0001; b, F = 62.055, P < 0.0001; c (left), F = 110.33, P < 0.0001; c (right), F = 91.663, P < 0.0001; d (left), F = 155.91, P < 0.0001; d (right), F = 61.985, P = 0.0001. Source data are provided as a Source Data file