Fig 2: Analysis of the association of HECTD1 expression levels with clinical outcome in cervical cancer. (A) Schematic representation of the HECTD1 gene and the positions of somatic mutations identified from The Cancer Genome Atlas. (B) Representative immunohistochemical images of HECTD1 (left panel) and SNAIL (right panel) protein expression levels in cervical cancer obtained from the HPA. (C) Low HECTD1 expression levels were associated with a poor survival of patients with cervical cancer. Kaplan-Meier plots presenting the relapse-free survival of cohorts of patients from the HPA. HECTD1 expression levels in patients is negatively correlated with SNAIL expression levels. HPA, Human Protein Atlas; HECTD1, HECT domain E3 ubiquitin ligase 1.

Fig 3: Subcellular localization of SNAIL. (A) CHX chase assay. HeLa cells were treated with 100 µg/ml CHX for the indicated time period and western blot analysis was performed with an anti-SNAIL antibody. Statistical analysis was performed using the Student's t-test. (B) Subcellular localization of SNAIL was analyzed using fluorescence microscopy in the Ctrl- or HECTD1-KD-transfected cells. The subcellular localization of SNAIL in individual cells is indicated with the arrow-line. Scale bar, 50 µm. (C) SNAIL nuclear signal intensities in Ctrl- and HECTD1-KD cells was examined by staining using anti-SNAIL antibodies. Data are represented as the means ± SD. **P<0.01 and >50 cells of each cell type was measured. (D) SNAIL nuclear signal in Ctrl- and HECTD1-KD cells with/without epidermal growth factor treatment was analyzed and the percentage of cells exhibiting high levels of nuclear signals are presented. Data are analyzed by one-way ANOVA and represented as the means ± SD. ***P<0.001. Ctrl, negative control; HECTD1, HECT domain E3 ubiquitin ligase 1; KD, knockdown; CHX, cycloheximide.

Fig 4: Regulation of HECTD1 expression levels by miR-210 under hypoxic conditions. (A) HeLa cells were treated with 500 µM CoCl2 to induce hypoxic conditions for the indicated time periods. RT-qPCR was performed to determine the expression levels of mature miR-21/210 and HECTD1. Each value was normalized to U6 expression levels and is expressed relative to the expression levels of miR-21/miR-210 in HeLa cells treated with the Ctrl. Data were analyzed by one-way ANOVA at 8 h and are presented as the means ± SEM of 3 independent experimental repeats. *P<0.05, ***P<0.001. Relative expression [log2 (treatment/control)] was quantified by normalizing values to expression levels recorded at 0 h. (B) Effect of the antagomir miR-21/210 transfection in HeLa cells on HECTD1 expression using RT-qPCR. HeLa cells were transfected with miR-21 or miR-210 antagomir or Ctrl for 16 h. Other cells were treated with 500 µM CoCl2 or 100 ng/ml EGF for 16 h. Expression levels were quantified by normalizing expression levels to cells not exposed to CoCl2 or EGF. Statistical analysis was performed using unpaired one-way ANOVA. ***P<0.001. RT-qPCR, reverse transcription-quantitative PCR; HECTD1, HECT domain E3 ubiquitin ligase 1; Ctrl, negative control; n.s., not significant; miR, microRNA; EGF, epidermal growth factor.

Fig 5: Interaction of HECTD1 with SNAIL. (A) In vivo interaction of HECTD1 with SNAIL. The interaction between HECTD1 and SNAIL expression levels were investigated using Co-Immunoprecipitation. HeLa cells were transfected with GFP-tagged SNAIL or GFP for 24 h, followed by sequential treatment with DMSO or 5 µM MG132 for 16 h. The cell lysates were immunoprecipitated with anti-GFP. IPs and WCLs were analyzed using western blotting to detect the expression levels of HECTD1 and GFP. (B) SNAIL ubiquitination assay. HeLa Ctrl and HECTD1-KD cells were transiently transfected with GFP-SNAIL or GFP empty vector for 24 h, followed by sequential treatment with DMSO or 5 µM MG132 for 16 h. The cell lysates were immunoprecipi-tated with anti-GFP antibody. IPs and WCLs were analyzed by western blot analysis with anti-ubiquitin, anti-GFP and anti-GAPDH antibodies. Results are representative of 2 experimental repeats. (C) HECTD1 promotes the ubiquitination of SNAIL in vivo. HeLa cells were transfected with expression plasmids for HECTD1 (Halo-HECTD1) and SNAIL (GFP-SNAIL) in the presence of 5 µM MG132 for 16 h. The cell lysates were immunoprecipitated with anti-GFP antibody and analyzed by western blot analysis with anti-ubiquitin antibodies. IP, immunoprecipitates; WCL, whole cell lysates; Ctrl, negative control; KD, knockdown; HECTD1, HECT domain E3 ubiquitin ligase 1.