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. 2018 Aug 3;9:857. doi: 10.3389/fphar.2018.00857

FIGURE 5.

FIGURE 5

Tp53 mutation may inhibit ubiquitination degradation of Siah1-mediated WISP1. (A) After lentivirus-mediated mouse Tp53R172H overexpression vector or control was stably transduced into SH-PAN cells, lentivirus-mediated human Tp53R175H or Tp53R273H overexpression vector or control was stably transduced into Capan-2 cells, Western blot analysis was performed to validate Siah1 and WISP1 levels. (B) Capan-2 cells were treated with 10 μM of Nutlin-3a at defined time period and were examined by immunoblotting. (C) Panc-1 cells transduced with lentivirus-mediated Siah1 silencing vector or control were treated with 100 μg/ml of cycloheximide (CHX) at defined time periods and were examined for WISP1 expression by Western blot. (D) His6-WISP1 was immobilized on Ni-NTA magnetic agarose beads (columns 2–4) and incubated with GST-Siah-1 (column 4), GST alone (column 3) or without additional protein (column 2). In column 1, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His6-tagged WISP1. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblotting using anti-GST and anti-WISP1 antibodies. In columns 5, 6, and 7, His6-WISP1, GST-Siah-1, and GST alone were run directly as input controls. (E) Myc-tagged WISP1, HA-tagged ubiquitin (Ub), and full-length Tp53 were transiently expressed in Capan-2 cells, as indicated, which were pre-treated with control or Siah-1 shRNA. After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by Western blot using anti-Myc antibody (upper) and anti-HA antibody (lower). White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image were corresponded to IgG heavy chains. (F) Simplified schematic diagram of the Tp53-Siah1-WISP1 pathway in PDAC cells (wild-type Tp53 or Tp53 mutation). Wild-type Tp53 may promote Siah1 protein levels, which may act as an E3 ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of WISP1. Meanwhile, Tp53 mutation may downregulate Siah1 protein levels, which may inhibit ubiquitination and degradation Siah1-dependent WISP1, and induce WISP1 nuclear import.