## 3 Results  
### TQ Induced UHRF1 Ubiquitination Related to HAUSP Downregulation in Human Cancer Cells  
The HAUSP deubiquitinase interacts with the UHRF1 protein and controls its ubiquitination status, thereby protecting UHRF1 from degradation by the proteasome [15,27]. TQ was shown to induce a sharp decrease in HAUSP in Jurkat cells [11]. We therefore investigated whether HAUSP is a main target of TQ in its mechanism of action leading to UHRF1 downregulation by analyzing gene expression after a 24-h exposure to TQ. The RNA-seq data showed that several deubiquitinases, including HAUSP, with known oncogenic proprieties were significantly downregulated in response to TQ treatment (Table 1). By contrast, two deubiquitinases, USP27X and USP35, which are known tumor suppressors, were insignificantly upregulated (Table 2).  
We then investigated whether UHRF1 ubiquitination is a common feature of TQ in cancer cells and whether this process involves the downregulation of HAUSP by evaluating the effects of TQ in two human cancer cell lines that highly express UHRF1: human colon cancer cells (HCT-116) [28,29] and Jurkat cells [1,30,31]. TQ induced a sharp decrease in the expression of UHRF1 and HAUSP at the protein level when administered at concentrations of at least 200 uM in HCT-116 cells (Figure 1A) and 30 uM in Jurkat cells (Figure 1B). The anti-proliferative effect of TQ in HCT-116 cells (Figure 1C) and in Jurkat cells (Figure 1D) was then analyzed under the same experimental conditions. Cell proliferation in response to TQ was decreased in a concentration-dependent manner in both cancer cell lines. TQ significantly inhibited cell proliferation starting from 30 uM in HCT-116 cells and the inhibition percentage reached approximately 50% at 100 uM, suggesting that TQ exerts its inhibitory effects on HCT-116 cells with an estimated half-maximal  
Figure 1: Dose-dependent effect of TQ on the protein expression of HAUSP and UHRF1 and cell proliferation. Human colon cancer (HCT-116) (**A**,**C**) and Jurkat cells (**B**,**D**) were exposed to increasing concentrations of TQ for 24 h. The expression of HAUSP and UHRF1 was analyzed by means of Western blotting, as described in the Materials and Methods. The cell proliferation rate was assessed via the WST-1 assay. Values are shown as means \(\pm\) S.E.M of three different experiments. * \(p\) < 0.05 versus control.  
Figure 2: Time course effect of TQ on HAUSP, UHRF1 and ubiquitinated UHRF1 and cell proliferation. Human colon cancer cells (HCT-116) (**A**,**C**) and Jurkat cells (**B**,**D**) were exposed to 200 \(\upmu\)M and 30 \(\upmu\)M TQ, respectively, for different times. The expression of HAUSP and UHRF1 was analyzed by means of Western blotting, as described in the Materials and Methods. The cell proliferation rate was assessed via the WST-1 assay. Values are shown as means \(\pm\) S.E.M of three different experiments. * \(p\) < 0.05 versus control.  
Taken together, these findings indicate that TQ induces a fast ubiquitination of UHRF1 in cancer cells that is related to HAUSP downregulation. This process could be an indispensable event in the subsequent degradation of UHRF1 and the upregulation of the p73 tumor suppressor and the inhibition of cell proliferation in response to TQ.