Computational protocol: Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells

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Protocol publication

[…] The protein search algorithm SEQUEST was used to identify unique protein peptides using the Proteome Discoverer data processing software (version 1.2, Thermo Fisher Scientific, Waltham, MA). The ratios of TMT reporter ion abundances in MS/MS spectra generated by HCD from raw data sets were used For TMT quantification. Fold changes in proteins between control and treatment were calculated as previously described []. [...] Proteins were extracted with RIPA buffer (1.5 M Tris pH 8.8, 1.75 g NaCl, 2 mL of 10% sodium dodecyl sulfate, 2 mL Triton X-100; all reagents from Fisher Scientific) plus a protease inhibitor cocktail. Protein quantification was performed with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). The samples absorbance was read and analyzed using the SoftMax Pro software (Molecular Devices, Sunnyvale, CA) coupled to a VersaMax Tunable microplate reader (Molecular Devices, Sunnyvale, CA) detecting at 562 nm excitation. Proteins were then analyzed by SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline and Tween 20 (TBST) and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this experiment were: anti-casp 12 (1:1000 dilution; Abcam, Cambridge, MA), anti-Ire1α (1:1000, Cell Signaling Technology), anti-GRP78 (1:1000, Cell Signaling Technology), anti-calreticulin (1:1000, Abcam), anti-PARP (1:1000, Cell Signaling Technology) and anti-LC3B (1:1000, Cell Signaling Technology). To confirm equal loading, blots were stained with 0.1% India ink. Horseradish peroxidase-conjugated secondary antibodies were used, and protein bands were visualized by a chemiluminescent method using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific, Waltham, MA). Images were analyzed with the ImageJ software (Bethesda, MD, USA) by comparing band densities (peak percent). […]

Pipeline specifications

Software tools SoftMax Pro, ImageJ
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Neoplasms, Prostatic Neoplasms
Chemicals Curcumin, Glucose, Inositol, Oxygen, Poly Adenosine Diphosphate Ribose