Fig. 4. The effect of the CBS-H₂S axis on the activation of VEGF and the effect of VEGF on the activation of CBS.

a, c Representative western blot images and relative band intensities of VEGF in SW480 WT and CBS-KD cells with or without GYY4137 treatment. GYY4137 (+) indicates that 50 μM GYY4137 was added, same as below. b, d Representative western blot images and relative band intensities of CBS in SW480 cells treated with VEGF with or without bevacizumab. VEGF (+) indicates that 30 ng/mL VEGF was added; bevacizumab (+) indicates that 3 μg/mL bevacizumab was added, as indicated below. e VEGF mRNA expression in SW480 WT and CBS-KD cells with or without GYY4137 treatment. f CBS mRNA expression in SW480 cells treated with VEGF with or without bevacizumab. g, h Schematic illustrations of the human VEGF gene promoter containing a putative AP-1 binding site and the human CBS gene promoter containing a putative NF-κB site. The PCR primer locations are also indicated. i AP-1 mRNA expression in SW480 WT and CBS-KD cells with or without GYY4137 treatment. j NF-κB mRNA expression in SW480 cells treated with VEGF with or without bevacizumab. k, m SW480 cells in different groups were subjected to immunofluorescence staining for AP-1 or NF-κB. l The percentage of AP-1 nuclear translocation. (N) The percentage of NF-κB nuclear translocation. o, q Representative western blot images and relative band intensities of AP-1 in the nucleus of SW480 WT and CBS-KD cells with or without GYY4137 treatment. p, r Representative western blot images and relative band intensities of NF-κB in the nucleus of SW480 cells treated with VEGF with or without bevacizumab. s ChIP analysis showed the binding of AP-1 to the promoter of VEGF in SW480 WT and CBS-KD cells with or without GYY4137 treatment. t ChIP analysis showed the binding of NF-κB to the promoter of CBS in SW480 cells treated with VEGF with or without bevacizumab. u Quantification of the fold change in VEGF promoter activity in SW480 WT and CBS-KD cells with or without GYY4137 treatment in luciferase reporter assays. A luciferase reporter plasmid containing the promoter region of VEGF was constructed (VEGF-luc). Cells were transfected with the VEGF-luc plasmid using Lipo3000 transfection reagent. v Quantification of the fold change in CBS promoter activity in SW480 cells treated with VEGF with or without bevacizumab in luciferase reporter assays. A luciferase reporter plasmid containing the promoter region of CBS was constructed (CBS-luc). Cells were transfected with the CBS-luc plasmid using Lipo3000 transfection reagent. (n = 3, biological replicates) * and # represent P < 0.05 vs. the control group.