5‐Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing β‐catenin

The following are the supplementary data related to this article:

Supplementary data

Supplementary Figure 1 5‐HT does not promote proliferation in all HCC cell lines. (A) The relative viability of five HCC cell lines cultured in 10% FBS, or SFM with or without 5‐HT (50 μM and 100 μM 5‐HT) was detected by MTT. (B) The relative viability of HuH‐7 and HepG2 cells cultured in 10% FBS with or without 5‐HT (50 μM and 100 μM 5‐HT) was detected by MTT.

Supplementary Figure 2 Affect of 5‐HT on various HCC characteristics. 5‐HT does not affect (A) cell migration as demonstrated by wound‐healing assay, (B) apoptosis, and (C) cell cycle as demonstrated by flow cytometry. 5‐HT inhibited (D) autophagy as shown by western blot of autophagy related protein markers, p62 and LC3B.

Supplementary Figure 3 5‐HT does not affect the Wnt/β‐catenin signalling in the presence of 10% FBS. HuH‐7 and HepG2 cells were cultured in 10% FBS with 50 μM 5‐HT for the indicated time points. Total β‐catenin, active β‐catenin, p‐β‐catenin, Axin1, p‐GSK3β, and total GSK3β protein levels remain unchanged.

Supplementary Figure 4 Wnt/β‐catenin pathway inhibitor, ICG‐001, attenuates HCC proliferation in the presence of 5‐HT. (A) ICG‐001 reduced cyclin D1 protein levels in HuH‐7 and HepG2 cell lines with and without the presence of 5‐HT. (B) The relative viability of HuH‐7 and HepG2 cells was detected by MTT. The cells were cultured in SFM and treated with ICG‐001 and/or 5‐HT. ICG‐001 inhibited HCC proliferation in both cell lines (ICG‐001 + 5‐HT vs 5‐HT, ***P < 0.001, two‐way ANOVA).

Supplementary Figure 5 IgG were used as negative control for Co‐IP detection in HuH‐7 cells.