Fig. 3: NINJ1 knockdown increased the stability, expression, and activity of xCT.

A–E Control and NINJ1-knockdown HT-1080 cells were fixed in 3.7% paraformaldehyde for 15 min. PLA was subsequently conducted to examine the interaction between xCT and NINJ1. Representative images for the (A) vector, (B) NINJ1-knockdown, (C) NINJ1 antibody alone, and (D) xCT antibody alone conditions, along with (E) quantification of PLA signals, are presented. Scale bar: 20 μm. F Co-IP and Western blot were used to detect the interaction between endogenous xCT and NINJ1-V5. NINJ1 was pulled down using the V5 tag, and the interaction with xCT was detected by Western blot. G xCT expression was increased following NINJ1 knockdown in HT-1080 cells, which was verified by Western blot. H-I Enhanced xCT protein stability post-NINJ1 knockdown in HT-1080 cells was validated by cycloheximide treatment and subsequent Western blot analysis. J Cystine uptake levels of control and NINJ1-knockdown HT-1080 cells following erastin treatment (10 μM, 4 h) were determined by Cystine Uptake Assay Kit. K Glutamate release levels of control and NINJ1-knockdown HT-1080 cells following erastin treatment (1.25 μM, 20 h) were determined by Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit. Error bars in (E) and (I–K) represent SEM (n = 15+ for E, n = 3+ for I–K).