Figure 1.
In vitro characterization of the triple color MDA-MB-231 cell line.
(1.A) Schematic representation of the lentiviral constructs used for developing triple color MDA-MB-231 cells. (1.B) Graph representing the emission spectrum of the three luciferases used in the study. ExtGluc in blue, CBG99 in green and PpyRE9 in red. (1.C) Graph representing the correlation between the CBG99 luciferase signal and cells number in the MDA-MB-231 cell line. (1.D) Graph represents the correspondence between bioluminescent signal from CBG99 luciferase expressing MDA-MB-231 and the absorbance signals of MTS assay generating a kill curve using puromycin.
Figure 2.
Time course analysis of TNFα-induced NF-κB activation in the triple color MDA-MB-231 cell line.
Non-significant differences were detected in NF-κB activation between 24, 48 and 72 hours of stimulation with TNFα (10 ng/ml).
Figure 3.
Celastrol has an inhibitory effect on TNFα-induced NF-κB signaling in MDA-MB-231 cells.
(3.A) Graph representing the decrease in fold NF-κB induction in MDA-MB-231 cells using celastrol by the multicolor assay (* p value <0.05; ** p value <0.01). (3.B) Composite image of the unmixed spectrum of luciferases: green signals represent CBG99 emission, red signals represent PpyRE9 emission spectrum and blue signals represent ExtGluc expression in control cells (first column) and cells treated with TNFα in combination with increasing concentrations of celastrol (0.1–3 µM). (3.C) Left: graph reporting the relative band intensity of p65 protein from the nuclear extract of control sample and samples treated with TNFα (10 ng/ml) or TNFα + celastrol (0.1 µM; 0.5 µM and 1 µM). Right: p65 protein detection in nuclear extracts and GAPDH used as control protein.
Figure 4.
Effects of chemopreventive natural compounds on NF-κB promoter activity.
Graphs representing the effect on NF-κB induction in MDA-MB-231 cells treated with different concentration of resveratrol (4.A), sulphoraphane (4.B), curcumin (4.C) and betulinic acid (4.D) (* p value <0.05; ** p value <0.01).
Figure 5.
In vivo monitoring of NF-κB signaling by bioluminescence imaging.
Growth curve of triple colored MDA-MB-231 cells implanted in the mammary fat pad of female athymic mice and measured by BLI (5.A). Red and green luciferase emission spectra as resulted from the spectral unmixing analysis applied to the series of acquired images (5.B). Representative picture of unmixed and composite images obtained with unmixing algorithm application at 0 and 24 hours after TNFα injection in a mouse model of breast cancer (5.C). On the left, the images corresponding to the green signal (vitality) while in the middle the images corresponding to the red signal (NF-κB induction). The graphs represent the average unmixed red and green signals obtained 0 and 24 hours after TNFα injection in three different mice. (5.D) Representative picture of unmixed images obtained with ex vivo analysis of tumors derived from mice challenged with or without TNFα. (5.E) Image showing Gluc expression on the membrane of cells derived from excised tumors and detected using a polyclonal anti-Gluc antibody and a FITC-conjugated secondary antibody.
Figure 6.
Celastrol has an inhibitory effect on TNFα-induced NF-κB signaling in breast cancer cells in vivo.
Graph reporting the red/green signal ratio as measured in the group treated with TNFα or TNFα + celastrol (2 mg/kg) at 0 and 24 hours. Treatment with celastrol lowers the red/green signal ratio demonstrating to act on NF-κB signaling.
Figure 7.
Effects of chemopreventive natural compounds on caspase 3/7-mediated apoptosis in vitro.
Graph representing caspase 3/7 activity measured with the caspase 3/7 glo assay and corrected for cell vitality at 4 hours after treatment with celastrol (7.A) resveratrol (7.B), sulphoraphane (7.C), curcumin (7.D) and betulinic acid (7.E).
Figure 8.
Celastrol induces caspase 3/7-mediated apoptosis in vivo.
Graph reporting the in vivo caspase 3/7 activity measured using DEVD-luciferin in control and treated groups at 0 hours, 4 hours and 24 hours. A single treatment of celastrol (2 mg/kg) significantly increased the fold induction of caspase 3/7 activity at 24 hours compared to controls (* p value <0.05; ** p value <0.01).