Fig 2: The effects of TFEB knockdown in PC12 cells. NC, si-TFEB#1, si-TFEB#2, and si-TFEB#3 were designed, synthesized, and transfected into PC12 cells. The transfection efficiency of the si-TFEBs were assessed by qRT-PCR (A) and Western blot (B). *P<0.05, **P<0.01 vs. NC group. TFEB, transcription factor EB; NC, negative control; si, small interfering; qRT-PCR, quantitative real-time polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 3: TFEB knockdown reversed the protective effect of AS-IV by mediating ferroptosis in H2O2-treated PC12. H2O2-damaged PC12 cells were treated with AS-IV, si-TFEBs, and/or FIN56. (A) Flow cytometry was performed to determine the rate of cell apoptosis in each group. (B) DCFH-DA was used to monitor ROS level in each group. Magnification, ×400; scale bar =20 µm. (C) TEM was used to observe the morphology of the mitochondria in each group. Scale bar =500 nm. (D) The levels of ferroptosis-associated proteins (SLC7A11 and GPX4) were measured by Western blotting analysis in each group. **P<0.01 vs. blank group; ##P<0.01 vs. H2O2 group; &P<0.05 vs. H2O2 + AS-IV + NC group; $P<0.05 vs. H2O2 + AS-IV + si-TFEB group; AS-IV; astragaloside IV; ROS, reactive oxygen species; DCFH-DA, dichloro-dihydro-fluorescein diacetate; TEM, transmission electron micrograph; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SLC7A11, solute carrier family 7 member 11; GPX4, glutathione peroxidase 4.

Fig 4: TFEB knockdown and ferroptosis promotion reversed the protective effect of AS-IV on cell injury induced by H2O2 in PC12 cells. H2O2-treated PC12 cells were incubated with 0.1 µM AS-IV, si-TFEBs, and/or 5 µM FIN56. (A) The viability of PC12 cells was monitored using the CCK-8 assay. (B) Flow cytometry was used to observe the changes in the rate of cell apoptosis in each group. (C) Caspase-3 activity was determined to analyze cell apoptosis. (D) The level of LDH was assess using ELISA. (E,F) IF assay (E) and Western blot (F) was conducted to determine the changes in TFEB expression in the nucleus and cytoplasm of PC12 cells. Magnification, ×400; scale bar =20 µm. *P<0.05, **P<0.01 vs. blank group; #P<0.05, ##P<0.01 vs. H2O2 group; &P<0.05 vs. H2O2 + AS-IV + NC group; $P<0.05 vs. H2O2 + AS-IV + si-TFEB group. TFEB, transcription factor EB; AS-IV; astragaloside IV; si, small interfering; CCK-8, cell counting kit 8; LDH, lactate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; IF, immunofluorescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 5: AS-IV protects PC12 cells against injury induced by H2O2 and enhances TFEB expression. PC12 cells were treated with 300 µmol/L H2O2, 0.1 µM AS-IV, and/or 5 µM FIN56. (A) The CCK-8 assay was used to determine the viability of PC12 cells; (B) Caspase-3 activity was measured to evaluate cell apoptosis; (C) ELISA was performed to verify the LDH concentration in PC12 cells; (D) flow cytometry was performed to determine the rate of apoptosis in PC12 cells. (E, F) TFEB expression was measured by using immunofluorescence (E) and Western blot (F), scale: 20 µm. *P<0.05, **P<0.01 vs. blank group; #P<0.05, ##P<0.01 vs. H2O2 group; AS-IV; astragaloside IV; CCK-8, cell counting kit 8; ELISA, enzyme-linked immunosorbent assay; LDH, lactate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.