Journal: Acta Pharmaceutica Sinica. B
Article Title: Inactivation of TFEB and NF- κ B by marchantin M alleviates the chemotherapy-driven pro-tumorigenic senescent secretion
Figure Lengend Snippet: Inactivation of TFEB and NF- κ B contributes to suppression of SASP by Mar-M. (A) Western blot analysis of the P65, the phspho-p65 (p-P65) in PC3/Doc cells treated with Mar-M. (B) Immunoblots of p-P65 state in nuclear and cytosolic fraction of PC3/Doc incubated with Mar-M for 1 or 5 days. GAPDH and Histone 3 (H3) were used as control. (C) The nuclear translocation of P65 was detected by confocal microscopy, LPS was used as positive control. (D) The nuclear and cytoplasmic fraction of TFEB and TFE3 in PC3/Doc cells and PC3 cells. (E) Western blot analysis of the TFEB and TFE3 in PC3/Doc cells treated with Mar-M. (F) Nuclear fraction of TFEB in PC3/Doc cells with Mar-M, thapsigargin (Tg) and Starvation (Sv). (G) Immunofluorescent staining of TFEB-GFP detected in 293T cells treated with Mar-M for 24 h and stained with DAPI (to visualize nuclei). Starvation and thapsigargin were used as positive controls for TFEB nuclear translation. (H) siRNA knockdown of TFEB in PC3/Doc cells was performed and analysed the IL-1α 、 IL-1β and IL-6 levels. (I) Quantification of IL-1α 、 IL-1β and IL-6 levels were shown after TFEB overexpression in 293T cell treated with Mar-M. Results are representative of three independent experiments. Data are mean ± SD, * P < 0.05, ** P < 0.01 and *** P < 0.001. Scale bar: 20 μm.
Article Snippet: Thapsigargin (Tg) was purchased from Selleck (Houston, TX, USA).
Techniques: Western Blot, Incubation, Translocation Assay, Confocal Microscopy, Positive Control, Staining, Over Expression