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Supplementary Materials and Methods
Detection of acidic vesicular organelles (AVO). Cells (1 x 105) were plated on cover slips in 6-well
plates and allowed to attach by overnight incubation. Following treatment with DMSO (control) or (-)gossypol, cells were stained with 1 µg/mL acridine orange in PBS for 15 min, washed with PBS, and
examined under a Olympus fluorescence microscope at 60 x magnification.
Supplemental Figure Legends
Figure S1. Immunoblot analysis of the protein levels of Bak and Bax in prostate cancer cell lines and
normal prostate epithelial cells (PrEC)
Figure S2. Prostate cancer cells were treated with 10µM (-)-gossypol or DMSO and pictures were
taken by phase-contrast microscopy at 24 h.
Figure S3. Prostate cancer cells were treated as indicated for 24h, fixed in 70% ethanol, stained with
propidium iodide (PI), and analyzed by flow cytometry for apoptosis (sub-G1).
Figure S4. Apoptosis induced by (-)-gossypol (with or without Z-VAD) in prostate cancer cells was
assayed by sub-G1 analysis, caspase-3 activity and PARP cleavage.
Figure S5. (-)-Gossypol preferentially induces autophagy in apoptosis-resistant prostate cancer cells
as revealed by acridine orange staining. A, (-)G-induced autophagy in prostate cancer cells as analyzed
by acridine orange staining. The cells were treated for 16h, then stained with acridine orange. Yellow
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arrows indicate the red acidic vesicular organelles (AVO) in autophagic cells. B, Quantitative data from
A, as percent of cells with orange punctates (50 cells in one field, n=5).
Figure S6. Expression level of Bcl-xL and Mcl-1 in CL-1 and PC-3 cells after treatment with
indicated dose of (-)-gossypol.
Figure S7. (-)-Gossypol mediated regulation of Beclin1 (A) and Bcl-2 (B). Immunoblotting of Beclin1
or Bcl-2 for CL-1 cells treated with 10µM (-)-gossypol, 10µM CHX or their combination at the
indicated time points. The values below each bands are relative protein levels from densitometric
analysis of the immunoblot bands, with the levels of DMSO control (time = 0h) set as 1.
Figure S8. (-)-gossypol induced autophagy in CL-1 xenograft tumors in vivo. Immunoblotting for
Beclin1, LC3 and Bcl-2 using the lysates from CL-1 xenograft tissues treated with vehicle control or (-)gossypol for 5 weeks.
Table S1. Human Autophagy Gene Expression PCR Array analysis identified genes whose expression
was up- and down-regulated by (-)-gossypol treatment of CL-1 cells. Shown is the list of genes whose
expression was significantly up-regulated or down-regulated following (-)-gossypol treatment for 24h,
as compared with the DMSO control.
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