Additional file 1 of MHY@MO@PPP inhibits ferroptosis positive feedback and delays the acute kidney injury-to-chronic kidney disease transition by remodeling the Fe2+-mTORC1-GPX4 axis

Note: (A) LC-MS/MS detection of the characteristic peak of MHY1485 in the MHY@MO@PPP nanozyme complex to verify the encapsulation of MHY1485; (B) LC-MS/MS detection of the release behavior of MHY1485 from the MHY@MO@PPP nanozyme complex under different pH conditions; (C) LC-MS/MS detection of the release behavior of MHY1485 from the MHY@MO@PPP nanozyme complex under different H2O2 stimulation conditions; (D) Laser scanning microscopy observation of the uptake of nanomaterials by HK-2 cells; red fluorescence represents Dil, and blue is DAPI nuclear staining (scale bar: 25 μm).

Figure S3. Construction and phenotypic analysis of the HK-2 cell ferroptosis model under a high iron environment. Note: (A) JC-1 fluorescence staining detected mitochondrial membrane potential changes in HK-2 cells, bar: 25 μm; (B) DCFH-DA fluorescence probe detected intracellular ROS generation levels in HK-2 cells, bar: 25 μm; (C) Biochemical kit detected intracellular MDA content in HK-2 cells; (D) Biochemical kit detected intracellular GSH content in HK-2 cells; (E) CCK-8 assay detected changes in HK-2 cell viability; (F) IF staining combined with laser confocal microscopy detected the expression and distribution of Fibronectin protein in HK-2 cells, bar: 25 μm.

Cell experiments were repeated three times.

• indicates comparison between two groups, p < 0.05, **p < 0.01, ***p < 0.001. Figure S4. Analysis of iron homeostasis dysregulation and cell death characteristics in the ferroptosis model. Note: (A-B) FerroOrange fluorescent staining detection of intracellular free iron levels in HK-2 cells (A) and quantitative analysis results (B), bar: 25 μm; (C) Flow cytometry Annexin V/PI staining detection of HK-2 cell death proportion; (D) Quantitative analysis of PI-positive cell distribution in HK-2 cells by flow cytometry. Cell experiments were repeated three times.
• indicates comparison between two groups, p < 0.05, ***p < 0.001. Figure S5. Analysis of molecular expression profiles and renal injury markers in the ferroptosis model. Note: (A) Western blot detection of ferroptosis-related proteins (GPX4, ACSL4, FTH1) expression in HK-2 cells; (B) Western blot detection of autophagy-related proteins p-mTOR and LC3II/I expression in HK-2 cells; (C) Western blot detection of oxidative stress-related protein SOD2 expression in HK-2 cells; (D) RT-qPCR detection of mRNA expression of renal tubular injury markers KIM1, NGAL, and CYSC in HK-2 cells; (E) RT-qPCR detection of mRNA expression of fibrosis and EMT-related genes ACTA2, Fn1, and VIMENTIN in HK-2 cells. Cellular experiments were repeated three times.
• indicates comparison between two groups, p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure S6. Effects of MHY@MO@PPP nanocomposites on iron ion homeostasis and ferroptosis in cells. Note: (A-B) FerroOrange detection of Fe2+ levels in HK-2 cells, bar: 25 μm; (C-D) Annexin V/PI flow cytometry detection of Annexin V/PI-positive cell death proportion. Cell experiments were repeated three times. Comparisons were analyzed by one-way ANOVA followed by Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. Figure S7. Detection of cellular Fe2+ load, death-related phenotypes, and renal injury/fibrosis markers after intervention of the Fe2+-mTORC1-GPX4 axis. Note: (A-B) FerroOrange detection of Fe2+ levels in HK-2 cells, scale bar: 25 μm; (C-D) Annexin V/PI flow cytometry detection of death-related Annexin V/PI-positive cells; (E) RT-qPCR detection of renal injury markers KIM1, NGAL, and CYSC; (F) RT-qPCR detection of fibrosis/EMT markers ACTA2, Fn1, and VIMENTIN. Cell experiments were repeated three times.
• indicates comparison between two groups, p < 0.05, **p < 0.01, ***p < 0.001.