Figure legends
Figure 1. 1a. Expression of SOD1, LDH 2, 3&4, and MMP2, MMP9 in human breast tumor (n=3) and its corresponding surrounding tissues (n=3): Lane distribution-1, 2, 3-surounding & 4, 5, 6 –Tumor.
1b. Activity assay of SULT1A1 in human tumor tissue (n=3) and its corresponding surrounding areas (n=3).
1c. SULT1E1 (protein) expression in tumor (n=6) tissue and their corresponding surrounding (n=6) tissue are calculated and densitometric data are presented. (blot image not shown). Level of significance in Student’s t –test is, b= p<0.01.
1d. HIF1α (protein) expression in tumor tissue (n=6) and their corresponding surrounding (n=4) tissue with densitometric analysis. Molecular weight marker β-galactosidase (120kD) was used in this study. Human β-actin was used to verify the control protein. Level of significance in Student’s t –test is, b= p<0.01.
1e. SULT1E1 (gene) expression RTPCR data in tumor (n=5) tissue and their corresponding surrounding (n=3) tissue are calculated and densitometric data are presented. Level of significance in Student’s t –test is, b= p<0.01.
Figure 2. Immunohistochemistry- SULT1E1 expression in tumor tissue and adjacent surrounding tissue. HE staining- Histoarchitecture of tumor and adjacent surrounding tissue.
Figure 3. 3a. Gel zymographic data of SOD activity in serum and liver tissue and GPx activities in liver tissues of rats treated with DAS, chalcone, or DAS+chalcone.
3b. Gel zymographic data of SOD and catalase activity in the liver tissues of rat treated for 3, 5 or 12 hours with DAS, chalcone or DAS+chalcone.
3c. Gel zymographic data of SOD and GPx activity in liver tissue of rats treated for 24, 48 and 72 hours with DAS, chalcone or DAS+chalcone.
Figure 4. 4a. Expression of rSULT1E1 protein and its densitometric analysis from rat treated with DAS, chalcone (Chal) or DAS+chalcone for 24, 48 and 72 hours and in one group DAS+chalcone with a second dose of DAS. Expression of rβ-actin was studied from all these experimental groups. Lane distribution- details are mentioned at the bottom of the lanes.
4b. Expression of rSULT1E1 protein and its densitometric analysis in rat liver cells (primary culture) treated with DAS, chalcone or DAS+chalcone for 3, 5 and 12 hours. Lane distribution- details are mentioned bottom of the lanes. Two molecular weight marker M1 and M2 were also run to verify SULT1E1 protein.
4c. Expression of rSULT1E1 mRNA from the liver of rat treated with DAS, chalcone or Das+chalcone and their densitometry data are presented. Expression of rβ-actin mRNA was also screened as a loading control.
Figure 5. Expression of rHIF1α protein and its densitometric analysis in rat model treated with DAS, chalcone (Chal) and DAS+chalcone for 24, 48 or 72 hours and a group with chalcone second dose. Lane distribution of -5a, As mentioned at the bottom of the blot image.
5b. Densitometric analysis and relative band densities of fig. 5a are presented as bar in the diagram.
5c. Gel zymographic data of MMP2 and MMP9 from the liver tissues of rat treated with DAS, chalcone or DAS + Chalcone for 7 days treatment. 5e. Gel zymographic data of MMP2 and MMP9 from the liver tissues of rat treated with DAS, chalcone or DAS + chalcone for 24, 48 or 72 hours.
Figure 6. Different parameters were measured from rat liver tissue after in-vitro treatment with DAS, chalcone or DAS+chalcone and data are presented as bar diagram. The representation of bars are mentioned in the figures-Individual data from in vitro experiments are presented in fig 6a-6c. Different groups had 3-5 animals per group as depicted in the picture and animals were serially sacrificed at stipulated time interval. In vivo experimental data from 7 days treatment groups are presented in fig 6d-6g. Bars represent the means ± SE from 4 animals in each group. Levels of significances of difference between two groups are verified Student’s t test.
Figure 7 Schematic representation of breast cancer association with redox-regulated SULT1E1 dysfunction and HIF1a/MMPs up-regulations. The prominent therapeutic role of Dialylsulfide (DAS) and chalcone via reversal of these protein dysfunctions has been summarized.
Table 1. The protein density and mRNA density (in surrounding and tumor) from western blot and RT-PCR data