Fig. 5: Docetaxel enhances TFEB activity through ROS generation.

a AGS cells were treated with docetaxel (10 nM) with or without NAC (5 mM) for 12 h followed by incubation with CM-H2DCFDA. The cells’ fluorescence intensity was then measured using flow cytometry. *P < 0.05. b As described in (a), nuclear and cytosolic fractions from both control and docetaxel-treated cells were probed for TFEB and then quantified. The same membrane was then stripped and reprobed for α-tubulin or Lamin AC as loading control. c AGS cells were transiently transfected with the TFEB-luc reporter construct. After 24 h, the cells were treated with docetaxel (10 nM) for another 12 h with or without NAC (5 mM) and the relative luciferase activity was measured. RLU refers to relative luciferase units. *P < 0.05. d As in (a), after treatment, AGS cells were harvested for RNA extraction. Real-time PCR was used to determine mRNA level changes in known TFEB target genes. GAPDH served as a loading control. All values are means ± SD at least three independent experiments. Student’s t test, *P < 0.05