Research Paper Volume 15, Issue 20 pp 11033—11051

Figure 4. The oncogenic activity of SSH1 is mediated by circadian rhythm disruption and Wnt/β-catenin signaling activation. (A) Dot plots show the correlation between CLOCK and SSH1 expression (left), or BMAL1 and SSH1 expression (right). (B) The protein–protein interaction network constructed by the STRING database shows the interaction between SSH1, ‛actin cytoskeleton builders’, and ‛circadian oscillators’. (C) Heatmap of the expression profiles of VIM, CTNNB1, GRB2, LIMK1, ACTB, CFL1, NPAS2, CRY1, ARNTL, SSH1, SNAI1, LRP6, WNT3, CXCR4, CXCL12, FGF1, FGFR1, GSN, BHLHE40, NR1D1, RORA, PER1, and CLOCK. (D) Representative western blot images showing the effect of 0 - 20 μM SenA or Huh7-SSH1^-/- on the protein expression of SSH1, CFL1/2, CLOCK, BMAL1, CRY1, WNT3, β-catenin, LRP5/6, BCL2, VIM, and Snail. GAPDH was loading control. (E) Representative immunofluorescence staining images showing the expression and localization of SSH1, BMAL1, and β-catenin in Huh-WT, Huh7-SSH1^-/- and 20 μM SenA -treated Huh7 cells. DAPI stained for nuclear localization. (F) Representative IP western images showing the presence of BMAL1, SSH1, or β-catenin in IP complex of respective antisera on probing with SSH1 antisera in Huh-WT cells but not in the Huh-SSH1^-/- cells.