Figure 7.

NF-κBregulation in MDA-MB-231 cells upon ST08 treatment: (A) Western blot analysis NF-κB protein and its targets was done on ST08 treated MDA-MB-231 cell lysates. Each experiment was done in duplicates, and a representative image is shown for each marker. Quantification was done for each marker (B) and is represented as a bar graph of mean +/- SEM. One sample t-test and one way ANOVA test was performed, and the p-value was calculated between control, and ST08 treated groups (*: p-value < 0.05, **: p-value < 0.005). (C) ST08 induced upregulation of miRs like miR-215-5p,miR-26a/b,miR-451a/b,miR-199a,miR-708a-5p and miR-514a-3p which directly or indirectly inhibited NF-κB. Whereas miRs like miR-20a, miR-668, miR-30e, miR-210-3p were downregulated, which upregulated IKB, SOCS1, and indirectly downregulated NF-κB.Bar graph depicting log2 Fold change of miRNA (D) and mRNA (E) involved in NF-κB pathway. (F) miRNA-mRNA regulatory network of NF-κB pathway upon ST08 treatment in MDA-MB-231 cells. ST08 regulates TFs like ZNF843, ZNF143, and EGF3 to regulate the expression of miR-708a-5p. miR-708a-5p,miR-514a-3p regulates NF-κB. ST08 also regulates expression of other miRs like miR-411-3p,miR-26b-3p/5p, miR-205-5p,let-7f-5p, miR-576-5p, miR-148b-3p,miR-125a-5p and miR-202-5p. These miRs regulate the expression of their targets, as shown in the network. (G) Effect of ST08 and IKK-16 on PMA-induced proliferation in MDA-MB-231 cells as tested by LDH assay. (H) Bar graph depicting cell viability upon IKK-16 treatment for 48h on MDA-MB-231 breast cancer cell line as tested by LDH assay. Experiments were performed a minimum of three times, and the bar graph shows mean ± SEM. One-way ANOVA test was performed and p-value was calculated between control and treated groups (*p < 0.05, **p < 0.005, ***p < 0.0001, ns, not significant).