I am checking the apoptosis-inducing effect of a drug on a colon cancer cell line.
I have to check the apoptotic pathway proteins by western blot.
Please suggest which main proteins I should check for confirmation of apoptosis.
I have checked the caspase-3 and PARP already.
Cleaved caspase-3 doesn't necessarily indicate apoptosis as the cleaved version of caspase-3 binds XIAP very tightly. Thus, cleaved caspase-3 is not necessarily active, unless XIAP is also removed. You normally won't have to worry much about this, as XIAP undergoes auto-ubiquitilation and subsequent degradation upon release of SMAC from the mitochondria and can also be cleaved by calpains during apoptosis, but there are conditions whereunder intact XIAP can delay apoptosis, even after caspase-3 cleavage has occurred (see, for example, my paper about G-CSF-induced delay of neutrophil apoptosis).
Caspases-8 and -9 have to be dimerized in order to gain activation. Their cleavage stabilizes the dimer, but is not necessarily an indication of their activation. Caspase-8, for example, can be cleaved by caspase-6 in the absence of apoptosis induction.
Cleaved PARP is generally a good indicator of apoptosis and easy to detect by Western blot. p70S6K may be even more reliable. It's usually best to do a cell death assay to accompany your Western blot and check for multiple markers.
Check that p70S6K is cleaved completely to make sure the cells are dead. Caspase-3 activation and PARP cleavage does not mean the cells are dead. Ref:
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
More p70S6K related data in the paper below
https://www.researchgate.net/publication/292615481_Mitochondrial_oligomers_boost_glycolysis_in_cancer_stem_cells_to_facilitate_blebbishield-mediated_transformation_after_apoptosis
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
Article Mitochondrial oligomers boost glycolysis in cancer stem cell...
BIM or any Bcl-2 family as suggested by Ashok.
http://www.ncbi.nlm.nih.gov/pubmed/9430630
Caspases cleavage is a good indication that cells die by apoptosis. Study of Bcl-2 familly protein levels by western blotting is more hardly interpretable.
PARP, caspase-3, in lymphocytes you can also analyse Mcl-1 expression
You can also do TUNEL (terminal nick end labeling). If this stain is positive it means you have fragmentation of DNA and the cell is committed to apoptosis.
The specifc marker for apoptosis is activation of Caspase-3. If expression of cleaved caspase-3 in increased, then it will be surely apoptosis. Cleavage of caspase-3 is well established marker for apoptotic cell death. Modulation of Bcl-2 family proteins is associated with apoptosis but these are not markers for apoptosis
Cleaved caspase-3 doesn't necessarily indicate apoptosis as the cleaved version of caspase-3 binds XIAP very tightly. Thus, cleaved caspase-3 is not necessarily active, unless XIAP is also removed. You normally won't have to worry much about this, as XIAP undergoes auto-ubiquitilation and subsequent degradation upon release of SMAC from the mitochondria and can also be cleaved by calpains during apoptosis, but there are conditions whereunder intact XIAP can delay apoptosis, even after caspase-3 cleavage has occurred (see, for example, my paper about G-CSF-induced delay of neutrophil apoptosis).
Caspases-8 and -9 have to be dimerized in order to gain activation. Their cleavage stabilizes the dimer, but is not necessarily an indication of their activation. Caspase-8, for example, can be cleaved by caspase-6 in the absence of apoptosis induction.
Cleaved PARP is generally a good indicator of apoptosis and easy to detect by Western blot. p70S6K may be even more reliable. It's usually best to do a cell death assay to accompany your Western blot and check for multiple markers.
All the suggestion were well accepted mentioned by the above researchers, But there are caspases independent apoptosis factors also. There are some isoenzyme Apoptosis inducing factor 1 family is a mitochondrial protein which regulates apoptosis through BAT3 gene activation. Might you can check the BAT3 gene expression apart from cleaved caspases which is widely check at first instance for cell death experiment.
WB of Bcl-2, Bax, Bid, Bim recommend together with Apoptosis Assay Kit from Roche.
Bcl-2 protein family members such as Mcl-1, Bcl-2 and Bax. Recommend together with FACS (AnnexinV) and/or caspase 3/7 assay from Promega.
The "World Consensual Recepice" for Cell Death Analyses are "summarized" in the three attached articles by Galluzzy and colleagues.
Best regards
Robert
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