Image lab 4.1 analysis software 4

The MHPCs were harvested to perform western blotting analysis in cytoplasmic and nuclear extracts for NF-κB p65 (F-6; Santa Cruz Biotechnology Inc., TX, USA), p-NF-κB (p65 S529) (Invitrogen™), phospho-IKB-α Ser32/36 (5A5; Cell Signaling, EMD Millipore, MA, USA) and bcl-2 (C-2; Santa Cruz), as described in supplementary methods 1. Cytoplasmic and nuclear protein levels were normalized to β-actin C4; Santa Cruz) and histone 1 (AE-4; Santa Cruz), respectively. Protein levels were quantified by Gel imaging system (BIO-RAD, CA, USA), and expression levels were estimated by Image Lab 4.1 analysis software 4, BIO-RAD).

Total cytoplasmic and nuclear protein expression levels of the cultured HHPC and HHK were determined by western blot analysis, as we previously described [10 (link)]. We used NF-κB (p65) (F-6; Santa Cruz), phospho-NF-κB (p65 S529) (44-711G; Invitrogen^TM, Thermo Fisher Scientific), phospho-IKB-α Ser32/36 (5A5; Cell Signaling), and bcl-2 (C-2; Santa Cruz). We also used β-actin (C4; Santa-Cruz), for cytoplasmic and nuclear extract normalization. Protein levels were quantified by Gel-imaging system (BIO-RAD), in each nuclear and cytoplasmic cellular compartment, and expression levels were estimated by Image Lab 4.1 analysis software 4, BIO-RAD). PARP control was omitted as we focused on NF-κB induced oncogenicity rather than apoptosis.

We performed a statistical analysis using Graph Pad Prism 6 software. Data obtained from AQUA and real time qPCR analysis were calculated and analyzed by HistoRX^® (HistoRX^® Inc. CT, USA) and CFX96^TM (Bio Rad) software, respectively. To obtain differences of AQUA scores, thickness of mucosa or mRNA expression values between experimental and control groups we performed comparisons using ONE-WAY ANOVA, non-parametric Kruskal-Wallis or Friedman test and Dunn's multiple comparison test (significance was defined as P-values < 0.05). The correlation coefficient (r) between protein or mRNA expression levels of different groups was estimated by Pearson correlation (significance P values < 0.05). A Z-test was used to estimate significance of protein or gene expression differences between experimental and control groups that were estimated by Image Lab 4.1 analysis software 4, or CFX96TM (BIO-RAD, CA, USA).