Fig 1: Cell death mechanisms following LTP treatment vary between cell types. Following treatment with LTP, 1 mM H2O2, or 1 µM staurosporine, protein lysates were harvested. (A) BPH-1 and (B) PC-3 cell line lysates were probed for apoptosis (C-PARP) by western blotting. (C) Normal and (D) cancer primary epithelial lysates were probed for apoptosis (C-PARP) and also autophagy (LC3B I/II). ß-Actin was used as a loading control throughout. Band intensity quantification was performed using the ImageJ software. Further analysis of apoptotic activity was conducted in (E) primary epithelial cells using Caspase-glo 3/7 assay (Promega). Immediately following treatment, caspase-glo 3/7 detection reagent was added to all wells, and luminescence intensity was quantified at 24 h. Readings were normalised to untreated control and are expressed as mean±s.e.
Fig 2: p53-R9 induces apoptosis of human cancer cells in the presence or absence of wild-type human TP53.A Immunoblot of U2OS (TP53-/-) cell lysates after transfection with green fluorescent protein (GFP)-tagged plasmids show expression of GFP, GFP-p53-R9, and GFP-e. p53 at predicted sizes. Blot was probed with antibody to GFP. Bands lower than 47 kDa in p53-R9 lane are likely cleavage products or non-specific bands, indicated by *. B Human cancer cells (U2OS and HCT116) were transfected and sorted for expression of the fluorescent protein GFP, then analyzed for Caspase 3/7 activity using the Promega Caspase-Glo 3/7 assay, values were normalized to cell viability (Cell Titer Glo). p < 0.01 for both p53-R9 and elephant p53 v GFP; One way ANOVA v GFP with Dunnett’s multiple comparison test and Dunnet’s correction. C U2OS TP53-/- cells were transfected with plasmids encoding GFP-tagged proteins and sorted for expression of GFP. Caspase 3/7 activity was measured using the Promega Caspase-Glo 3/7 assay, values were normalized to cell viability (Cell Titer Glo). p < 0.0001 for both p53-R9 and elephant p53 v GFP; One way ANOVA with Dunnett’s multiple comparison test. D U2OS TP53-/- cells expressing mCherry were analyzed for quantity of Annexin V green positive cells. Area under the curve (AUC) was calculated for N = 3 experiments; Welch’s t test comparing AUCs, *p < 0.01 p53-R9 v p53-R9 + Z-VAD-FMK. Error bars represent SD of three images/well taken in three wells of a representative experiment.
Supplier Page from Promega for Caspase-Glo 3/7 Assay