Skip to main content
. 2024 Jul 3;121(28):e2320655121. doi: 10.1073/pnas.2320655121

Fig. 5.

Fig. 5.

Cystine deprivation regulates KCTD10- and USP18-mediated ubiquitylation of SLC7A11. (A) BT474 cells were cultured with cystine-free medium for different time points, followed by immunoblotting. (B) BT549 cells were cultured with indicated percentage of cystine for 24 h, followed by immunoblotting. (C) BT474 cells were cultured with cystine-free medium for different time points, followed by cystine addition at 24 h time-point for different time points, followed by immunoblotting. (D) SK-BR-3 cells were transfected with FLAG-KCTD10 plasmid, along with the vector control, cultured in cystine-containing or cystine-free media for 24 h, followed by IP-IB analysis of the indicated proteins. (E) HEK293 cells were transfected with indicated plasmids, cultured in cystine-containing or cystine-free media for 24 h, followed by Ni-beads pulldown, immunoblotting for SLC7A11. (F) SK-BR-3 cells were transfected with FLAG-USP18 plasmid, along with the vector control, cultured in cystine-containing or cystine-free media for 24 h, followed by IP-IB analysis of the indicated proteins. (G) HEK293 cells were transfected with indicated plasmids, cultured in cystine-containing or cystine-free media for 24 h, followed by Ni-beads pulldown, immunoblotting for SLC7A11. (H) BT474 cells were transfected with FLAG-KCTD10 plasmid, along with the vector control for 48 h, then cultured with cystine-free medium for different time points, followed by immunoblotting. (I) BT474 cells were transfected with siRNAs (si-NC or si-USP18s) for 48 h, then cultured with cystine-free medium for different time points, followed by immunoblotting. (J and K) MDA-MB-231 cells were transfected with indicated siRNAs and cultured with medium containing 100% and 3.125% cystine, respectively, then cell viability (J) and clonogenic survival (K) were measured (mean ± SD, n = 3). (L) MDA-MB-231 cells were transfected with indicated siRNAs and plasmids, cultured with medium containing 100% and 3.125% cystine, respectively, for 48 h, then cell viability was measured (mean ± SD, n = 3).