Fig. 1 Evaluation of H2O2-induced oxidative stress cell model A：Oxidative stress state of HEK293T cells treated with different concentrations of H2O2 was detected by DCFH-DA. B：Image J software for analyzing correlation between mean fluorescence intensity and H2O2 concentration in Fig. A

Fig. 2 Mito-OS-Timer targets to monitor the dynamic changes of mitochondrial oxidative stress induced by H2O2 in cell models A：Mito-OS-Timer，namely pLVX-Mito-OS-Timer recombinant expression plasmid map. B：Evaluate the expression of HEK293T cells transfected with Mito-OS-Timer plasmid. The fluorescence protein expression was detected by inverted fluorescence microscope after induction by Doxycyclin. Bar：20 μm. C：Mito-OS-Timer targets to monitor the changes of mitochondrial oxidative stress induced by H2O2 in cell models. The concentrations of H2O2 in the 6 groups were 0，10，50，100，200 and 300 μmol/L，respectively. Light avoidance treatment for 24 h（Green：Green fluorescence. Red：Red fluorescence. Merge：Red-green fluorescence superposition. Bar：20 μm）. D：Image J software for analyzing correlation between H2O2-induced concentration and changes in intracellular red-green fluorescence ratio in Fig. C

Fig. 3 Evaluation of Mito-OS-Timer system in rotenone-induced mitochondrial oxidation cell model A：Evaluation of targeted monitoring effect of Mito-OS-Timer based on rotenone-induced oxidative stress cell model Doxycyclin-induced HEK293T cells were treated with rotenone at different concentrations of 0，0.01，0.1，0.5，1 and 5 μmol/L for 24 h（Green：green fluorescence；Red：red fluorescence；Merge：red-green fluorescence superposition；Bar：20 μm）；B：Image J software analysis of correlation between rotenone induced concentration and intracellular red-green fluorescence ratio in Fig. A；C：Statistical results of cell count of HEK293T cells treated with different concentrations of rotenone

Fig. 4 Flow cytometry results of rotenone-induced mitochondrial oxidation in rotenone-induced cell models monitored by Mito-OS-Timer A：Fluorescence expression of rotenone treated cells was detected by flow cytometry. HEK293T cells were treated with 0，0.01，0.1，0.5，1 and 5 μmol/L rotenone for 24 h，and the fluorescence intensity was detected by flow cytometry；B：Image J software analysis the correlation between rotenone induced concentration in Fig. A and changes in intracellular red-green fluorescence ratio

Fig. 5 Relationship between FLCN gene expression and mitochondrial oxidative stress dynamic changes detected by Mito-OS-Timer A：pLVX-shFLCN recombinant plasmid map；B：1% agarose gel electrophoresis detection map（M：DNA Marker；1：FLCN gene fragment digested by EcoR I /Xho I，1 740 bp；2：pLVX-shRNA vector fragment digested by EcoR I /Xho I，7 881 bp；3：pLVX-shFLCN plasmid DNA，9621 bp）；C：FLCN gene expression in transfected cells was detected by western blot（WT：Wide type，FLCN gene expression in normal HEK293T cells；OE：overexpression，FLCN overexpression in HEK293T cells transfected with pcDNA-3HA-FLCN）；D：Mito-OS-Timer was used to detect dynamic changes of mitochondrial oxidative stress in OE，FLCN silencing（RNAi）and 0.1 μmol/L rotenone-induced（OS）cells（Green：green fluorescence；Red：red fluorescence；Merge：red-green fluorescence superposition；Bar：20 μm）；E：Flow cytometry was used to detect Mito-OS-Timer to monitor the cells；F：Quantitative analysis of flow cytometry results for the changes of red-green fluorescence ratio in various cells