Fig 2: Impact of resveratrol or/and ASO against ß1-integrin or against NF-?B on TME-promoted activation of metastasis- and apoptosis-linked biomarkers in CRC cells. Serum-starved RKO (A) and HCT116 (B) CRC cells, grown in 3D-alginate and differently treated for 10–14 days [untreated (Ba.Co.), TME control, resveratrol (1, 5 µM), ß1-SO/ASO (0.5 µM), NF-?B-SO/ASO (0.5 µM) or combination of 0.5 µM SO/ASO and resveratrol (5 µM); x-axis] were investigated with antibodies against pan-NF-?B, p65-NF-?B, FAK, p-FAK, MMP-9, CXCR4 and cleaved caspase-3. ß-actin served as loading control and statistical significance is shown by: *p < 0.05 and **p < 0.01 compared with the TME control. Y-axis: Densitometric units (y-axis) complementing Western blot results.

Fig 3: hCXCR4 expression was increased by Nrf2 overexpression.A–D WiL2-NS cells were co-transfected with pEZX-PG02-hCXCR4-gaussia luciferase (Gluc) plasmid DNA with pcDNA3.1 control or pcDNA3.1-Nrf2 plasmids using Viafect™. 30 h after transfection, Gluc activity of hCXCR4 promoter (pmt) was measured by using luminometer (A). RNA was extracted by using Nucleozol. mRNA of hCXCR4 was measured by RT-PCR (B) or qPCR (C). Cell lysates were prepared and the protein level of each protein was measured by using western blot analysis (D). E WiL2-NS cells were co-transfected with wild or mutant type of pEZx-PG02-hCXCR4-Gluc promoter plasmid DNA with pcDNA3.1 control or pcDNA3.1-Nrf2 plasmids using Viafect™. 30 h after transfection, WiL2-NS cells were incubated under hypoxic condition and Gluc activity was measured by using luminometer. Each experiment was performed at least five times. Data in a bar graph represented the means ± SD. *p < 0.05, **p < 0.01; significantly different from pcDNA3.1-transfected control group. #p < 0.05, ##p < 0.01; significantly different from control transfected with wildtype CXCR4 pmt plasmid (A, C, E).

Fig 4: CXCR4 expression was inhibited by the treatment with N-acetyl-l-cysteine (NAC).A–F WiL2-NS cells were incubated in the presence or absence of N-acetyl-l-cysteine (NAC) under hypoxic condition and treated with DCF–DA. ROS production was observed under fluorescence microscope (A) or it was measured by flow cytometry analysis (B). The mean fluorescence intensity (MFI) was analyzed by WinMDI 2.8 for ROS level and represented as bar graph (C). RNA was extracted by using Nucleozol. mRNA of hCXCR4 was measured by qPCR (D) or RT-PCR (E). Cell lysates were prepared and the protein level of each protein was measured by using western blot analysis (F). Each experiment was performed at least five times. Data in a bar graph represented the means ± SD. *p < 0.05, **p < 0.01; significantly different from control group under normoxic condition. #p < 0.05, ##p < 0.01; significantly different from NAC-untreated group (C, D).

Fig 5: Impact of resveratrol or/and ASO against ß1-integrin on TME-promoted activation of metastasis and apoptosis parameters in CRC cells. HCT116 (A) and RKO (B) derived from 3D-alginate cultures were grown untreated or treated with 5 µM resveratrol alone or in combination with 0.5 µM ß1-SO or 0.5 µM ß1-ASO (x-axis) and probed with antibodies against CXCR4, FAK, p-FAK, NF-?B, p65-NF-?B and cleaved caspase-3. Loading control: ß-actin. Densitometric units complementing Western blot results (y-axis). For densitometric analysis, data were compared to TME control: *p < 0.05 and **p < 0.01.