Fig 2: p65 activation enhances proliferation co-operatively with STAT-3. (A) Immunoblot showing expression of p65 after IL-6 treatment for 72 h. (B) Immunofluorescence image and relative fluorescence intensity of p65 in untreated and IL-6-treated cells (scale bar: 10 µm). The white arrow indicates p65. (C) Immunoblot showing expression of p65 in IL-6-treated or IL-6 plus p65-siRNA treated cells after 72 h of exposure. (D) Graph representing a shift in PI fluorescence in IL-6-treated or IL-6 plus p65-siRNA treated cells after 72 h. (E) Immunoblot showing expression of PCNA in IL-6-treated or IL-6 plus p65-siRNA treated cells after 72 h of treatment. (F) (i) Immunoblot showing expression of STAT-3 in IL-6-treated or IL-6 plus p65-siRNA treated cells after 72 h (ii) Immunoblot showing expression of STAT-3, p65, and PCNA in IL-6-treated or IL-6 plus Stattic exposed cells. (G) Co-immunoprecipitation analysis of STAT-3 with p65 after IL-6 exposure for 72 h The proteins were immunoprecipitated with anti-STAT-3 antibody and then probed with p65. *, *** and **** refers to p value significance of =0.1, =0.001 & = 0.0001 respectively.

Fig 3: IL-6-induced effects are suppressed by TGF-ß through inhibition of p65 and STAT-3. (A) Immunoblot showing expression of phosphorylated p65 after TGF-ß/IL-6 or TGF-ß plus IL-6 treatment for 72 h. (B) Immunofluorescence image and relative fluorescence intensity of p65 in TGF-ß/IL-6 or TGF-ß plus IL-6 treated cells (scale bar: 10 µm). White arrow indicates p65 protein. (C) Transcript levels of IL-6R after TGF-ß/IL-6 or TGF-ß plus IL-6 treatment for 72 h. (D) Immunoblot showing expression of STAT-3 in TGF-ß/IL-6 or TGF-ß plus IL-6 treated cells after 72 h of exposure. (E) Immunofluorescence image and relative fluorescence intensity of STAT-3 in TGF-ß/IL-6 or TGF-ß plus IL-6 treated cells (scale bar: 10 µm) White arrow indicates STAT-3 protein. (F) Immunoblot showing expression of SMAD2/3 after TGF-ß/IL-6 or TGF-ß plus IL-6 treatment for 72 h; immunofluorescence image and relative fluorescence intensity of SMAD2 in TGF-ß/IL-6 or TGF-ß plus IL-6 treated cells (scale bar: 10 µm). The white arrow indicates SMAD2 protein. **, *** and ns refers to p value significance of =0.01, =0.001 & not significant respectively.

Fig 4: SDF-1 inhibits RelA binding to CXCR4 promoter in vitro and in vivo.A Nuclear protein extracts were prepared from untreated EPCs and analyzed by EMSA with the CXCR4-biotin probe. Specific competition was performed with the 50-, 100- or 200-fold molar excess of unlabeled CXCR4 probe. B EPCs were pre-treated with FGF23 for 30 min followed by SDF-1 stimulation for 10 min. Treatment of NF-?B inhibitor (Helenalin) for 40 min was used as a positive control. Nuclear protein extracts were prepared and analyzed by EMSA with the CXCR4-biotin probe. C For supershift assay, RelA, p-RelA, or p50 Antibodies were incubated with untreated EPC nuclear extracts prior to the addition of the CXCR4-biotin probe. D For DAPA assay, untreated EPC nuclear extracts were incubated with the CXCR4-biotin probe and streptavidin beads. The beads were further analyzed by western blotting with RelA, p-RelA, or p50 Antibodies. E–G EPCs were pre-treated with FGF23 for 30 min followed by SDF-1 stimulation for 15 min. Incubation of NF-?B inhibitor (Helenalin) for 40 min was used as a positive control. For ChIP assays, cross-linked DNA were immunoprecipitated with RelA (E), p50 (F), or p-RelA (G) antibodies and subjected to PCR with CXCR4 specific primers. Treatment with IgG antibody was used as a negative control. PCR product of unprecipitated DNA was used as an input control.

Fig 5: SERPINB2 is associated with cell migration and apoptosis in KYSE150. A-B. Relative fold change of SERPINB2 (A) and RELA (B) 24 h after knockdown with antisense oligonucleotide (ASO) targeting SERPINB2. (C) Cell viability after ASO-mediated knockdown of SERPINB2. (D) Western blotting showing down-regulation of SERPINB2 and Caspase-3 after SERPINB2 knockdown. (E) Wound healing assay showing cell migration after SERPINB2 knockdown without EGCG treatment. (F,G) Statistical analysis for cell migration distance of three independent assays without (F) or with (G) EGCG. (H) Western blotting showing up-regulation of SERPINB2 and Caspase-3 after SERPINB2 overexpression. (I) Cell viability determined by CCK8 assay after SERPINB2 overexpression. Data are shown as mean ± SD. n = 3, *: p < 0.05; ***: p < 0.001, ns: not significant.