Fig 2: Complex IV subunit 4 isoform 2 (Cox4i2) depletion attenuated HHV7-induced ferroptosis. (A,B) Protein and relative mRNA levels of FTH, TFRC, SLC7A11, SLC3A2, and GPX4 were determined by Western Blotting and RT-qPCR in vivo. (C,D) Protein and relative mRNA levels of FTH, TFRC, IREB2, SLC7A11, SLC3A2, and GPX4 were determined by Western Blotting and RT-qPCR in HHV7-treated RSC96 cells with or without shCoc4i2. (E) Image of PGSK staining of RSC96 cells. Data are presented as the mean ± standard deviation, n = 3. Statistical significance was assessed with Student’s t-test and one-way ANOVA. **P < 0.01; *P < 0.05.

Fig 3: GPx4 inhibitors enhance antitumor effect of system xc− inhibitor in MTDH-high tumors in vivo.a, b Xenograft study using MDA-MB-231 cells expressing WT MTDH (high). This is a therapeutic study to test the effect of RSL3 on preexisting tumors. Mice were treated with either vehicle (ctrl, n = 4) or intratumoral RSL3 (100 mg/kg, 2 times per week, n = 5), or sorafenib via gavage (20 mg/kg, daily, n = 5), or the combination of RSL3 and sorafenib (n = 5) for 20 days. ***P < 0.001 by two-way ANOVA. a Tumor growth curve; b tumor weight after sacrifice. c A tumor prevention study using ML162 alone or in combination with sorafenib. Treatment was started 1 day after implantation of MDA-MB-231 cells expressing WT MTDH (high). ML162 was given by local injection at 50 mg/kg two times per week, and sorafenib was given orally daily. Treatment lasted for 2 weeks, followed by another week of observation before mice were sacrificed and tumors harvested. The combination of ML162 and sorafenib resulted in significantly smaller tumors compared to sorafenib alone (P = 0.047). *P < 0.05, **P < 0.01 by two-way ANOVA. d A scheme describing the role of MTDH in ferroptosis. Red or green color represents compounds and molecules/proteins that promote or inhibit ferroptosis, respectively. MTDH promotes ferroptosis by downregulating SLC3A2, GPX4, and GSH. All compounds (either in red or green) were tested in this study

Fig 4: Representative images of immunoreactivity for LAT1, 4F2hc, and Ki67 in human ATC and AG tissues. (a) immunoreactivity 0 for LAT1 in ATC tissues; (b) immunoreactivity 1 for LAT1 in ATC tissues; (c) immunoreactivity 2 for LAT1 in ATC tissues; (d) immunoreactivity 3 for LAT1 in ATC tissues. (e) immunoreactivity 0 for 4F2hc in AG tissues; (f) immunoreactivity 1 for 4F2hc in AG tissues; (g) immunoreactivity 2 for 4F2hc in ATC tissues; (h) immunoreactivity 3 for 4F2hc in ATC tissues. (i) immunoreactivity 0 for Ki67 in AG tissues; (j) immunoreactivity 1 for Ki67 in ATC tissues; (k) immunoreactivity 2 for Ki67 in ATC tissues; (l) immunoreactivity 3 for Ki67 in ATC tissues. (m) Cause-Specific Survival (CSS) rates of ATC patients according to LAT1 expression.

Fig 5: Detection of LAT1 and 4F2hc by western blotting and immunofluorescence analyses in 8505C, OCUT-2, and OCUT-6 human ATC cells. (A) Expression of LAT1 and 4F2hc levels in the indicated cells detected by western blotting. (B) The densitometry value of LAT1 was normalized α-tubulin. (C) The densitometry value of 4F2hc was normalized α-tubulin. (D) DAPI (4,6-diamidino-2-phenylindole is a blue fluorescent stain specific for DNA) nuclear staining was shown in blue. Both LAT1 (green) and 4F2hc immunoreactivities (red) were detected on the plasma membrane of 8505C, OCUT-2, or OCUT-6 cells. LAT1 (green) and 4F2hc immunostaining (red), and DAPI (blue) were merged and shown in 2D, indicating the coexistence of LAT1 with 4F2hc in the plasma membrane of 8505C, OCUT-2, or OCUT-6 cells, respectively.