Fig 2: CYP11A1 could regulate serum-free medium-induced cell mitophagy via changing cholesterol content. AGS cells were transfected with si-CYP11A1 or CYP11A1 plasmid, followed by treatment with AVT or cholesterol to counteract the impact of CYP11A1 manipulation on cholesterol. Then these cells were treated with a serum-free medium for 12 h. Next, the following procedures were performed in those cells. A-B Immunofluorescence staining of anti-Tom20 was performed to represent the total mitochondrial size, and quantification was done in the indicated groups. C-D Mitotracker-Red staining (C) represented the functional mitochondrial in the indicated groups, and quantification was done in the corresponding groups. E JC-1 staining represented the mitochondrial potential of the knockdown AGS cells without or with statin (Atorvastatin, AVT) treatment. F Co-staining of mitophagy dye and lysosomal dye was used to detect the mitophagy. G Electron microscopy showed representative mitophagy. H-I Examination of PINK1, Parkin, p62 and LC3i/ii by WB in the knockdown group alone or with AVT stimuli (H) and in overexpression cells alone or with cholesterol stimuli (I). J-K Examination of PINK1, Parkin, p62 and LC3i/ii by WB in AGS cells with CYP11A1 knockdown (J) or overexpression (K) under the infection of H. pyloriWT. M Mechanism diagram of this work. Generally, the interaction of CagA from H. pylori with CYP11A1 mediated the CYP11A1 redistribution outside the mitochondria and therefore caused mitochondrial cholesterol accumulation and subsequent mitophagy inhibition and tumor progression. Data and error bars were shown as mean ± SD of triplicate independent replicate experiments. For the assessment of data passing independence, normality, and homogeneity of variance, pairwise comparisons were conducted using one-way analysis of variance. Nonparametric tests were utilized in cases where the aforementioned conditions were not met. Significant flags and p-values are intricately linked in the following manner: (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001).

Fig 3: Helicobacter pylori induced the translocation of CYP11A1 protein out of mitochondria via CagA-CYP11A1 interaction and thus caused mitochondrial cholesterol accumulation. A-B Mitochondrial proteins (A) and cytoplasmic proteins (B) isolated from GC cells infected or uninfected with different H. pylori strains were subject to WB experiments to detect CYP11A1 and CagA. C Laser confocal microscopy revealed CYP11A1 protein expression and subcellular localization in uninfected HGC-27 cells and those infected with H. pyloriWT. D-E Mitotracker-Red and Filipin III co-staining represented mitochondrial cholesterol content in AGS cells either uninfected or infected with different H. pylori strains. F The mitochondrial cholesterol content represented by Mitotracker-red and Filipin III co-staining varied in represented AGS cells with either CYP11A1 manipulation or H. pyloriWT infection. G Colocalization analysis of F was performed using Plot Profile of image J software.

Fig 4: The CagA protein could directly bind to CYP11A1 protein. A The HDOCK protein docking database predicted the binding of CagA and CYP11A1. B-E Co-immunoprecipitation (Co-ip) assays were performed in different GC cells with or without H. pylori infection. AGS cells or HGC-27 cells were infected with different H. pylori strains alone or in combination with different H. pylori strains. Then the corresponding protein solutions were extracted and subjected to electrophoresis for the detection of the relevant molecules after immunoprecipitation with anti-CagA or anti-CYP11A1 magnetic beads. F-G Co-ip was performed in GC cells transfected with CagA plasmid or infected with CagA-positive H. pylori strains. H GST pull-down was performed to confirm the direct binding between CagA and CYP11A1. I Immunofluorescence staining (IF) of CagA and CYP11A1 was performed in GC cells and those infected by H. pyloriWT. J Representative images of Immunofluorescence for CagA and CYP11A1 in subcutaneous tumors tissue constructed from HGC27 cells alone and those infected by different H. pylori strains.

Fig 5: H. pylori disrupted the negative regulatory relationships between CYP11A1 and CYP19A1 in a CagA-dependent manner. A The string database predicted a strong correlation between CYP11A1 and CYP19A1. B The effects of different CYP11A1 levels on CYP19A1 in AGS cells alone or pretreated by various H. pylori strains infection by PCR. C The influence of CYP19A1 on CYP11A1 in AGS cells alone or infected by different H. pylori strains by PCR. D The effects of CYP11A1 on CYP19A1 in AGS cells or those infected with different H. pylori strains by WB. E The effects of CYP19A1 on CYP11A1 in AGS cells or those infected with different H. pylori strains by WB. F Luciferase reporter assay was conducted in AGS cells infected by different H. pylori strains. G Luciferase reporter assay was performed in AGS cells transfected with indicated plasmids for 48 h. Data and error bars were shown as mean ± SD of triplicate independent replicate experiments. For the assessment of data passing independence, normality, and homogeneity of variance, the Student's t-test was employed to compare the differences between the two sets of data. A mixed-design analysis of variance or one-way analysis of variance was used for pairwise comparisons. Nonparametric tests were utilized in cases where the aforementioned conditions were not met. Significant flags and p-values are intricately linked in the following manner: (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001).