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Fig 6 - Sph-mediated mtDNA release and autophagy-dependent ferroptosis contribute to Staphylococcus aureus-induced mastitis Yunfei Wei (9412265) Xinyu Zhao (102214) Ziyi Yin (4117480) Yanmin Zhang (391964) Yaxin Zhai (2562517) Baihe Zhan (25138380) Yeling Wang (371383) Jiakang He (16829817) Jianfeng Wang (232744) Xuming Deng (158166) Lei Xu (78676) Haihua Feng (198813) Biochemistry Microbiology Cell Biology Genetics Molecular Biology Environmental Sciences not elsewhere classified Immunology Biological Sciences not elsewhere classified Science Policy Infectious Diseases Virology mitochondrial contact site dysregulated inflammatory response disrupt mammary tissues potential therapeutic target key virulence determinant mastitis remain elusive dependent ferroptosis contribute >- induced mastitis >- induced ferroptosis sting signaling pathway mediated mtdna release cgas ), leading staphylococcus aureus </ promote gpx4 degradation aureus </ virulence factors sting axis potential role lysosome pathway underlying mechanisms thereby eliciting sphingomyelin phosphodiesterase organizing system micos ). major pathogen diverse arsenal cyclic gmp core component amp synthase >; however S. aureus Sph activates STING to promote autophagy-lysosome-dependent GPX4 degradation and ferroptosis. (A) Expression of GPX4 protein in HC11 cells treated with 32 μg/mL recombinant Sph and C-176 (5 μM) for 6 h. (B) Expression of of LC3B-I and LC3B-II levels in HC11 cells treated with recombinant Sph (32 μg/mL) for 6 h. (C) GPX4 expression in HC11 cells treated with recombinant Sph (32 μg/mL) alone or in combination with NH4Cl (10 mM) or chloroquine (20 μM) for 6 h. (D) Expression of GPX4 protein in HC11 cells treated with 32 μg/mL recombinant Sph alone or in combination with 3-MA (5 mM) or bafilomycin A1 (100 nM) for 6 h. (E-G) Representative confocal images and corresponding colocalization analyses of STING with GPX4 (E), LC3B with GPX4 (F), and LAMP2B with GPX4 (G) in HC11 cells treated with recombinant Sph (32 μg/mL), cGAMP (2 μg/mL), or C-176 (5 μM), as indicated, for 6 h. Scale bars, 10 μm. (H, I) Reciprocal co-immunoprecipitation analysis of Flag-GPX4 and Myc-STING in co-transfected NIH/3T3 cells using anti-Flag (H) or anti-Myc (I) antibodies. (J) Ratiometric imaging of Golgi luminal pH in NIH/3T3 cells expressing MGAT2-RpHLuorin2 following treatment with cGAMP (2 μg/mL) or bafilomycin A1 (100 nM). The 405/488 nm fluorescence ratio was used as an indicator of Golgi pH (MGAT2-RpHLuorin2). Data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ns, not significant. 2026-09-29T18:02:15Z Image Figure 10.1371/journal.ppat.1014644.g006 https://figshare.com/articles/figure/Fig_6_-_p_p_/34026603 CC BY 4.0