Journal: International Journal of Oncology

Article Title: HECTD1 regulates the expression of SNAIL: Implications for epithelial-mesenchymal transition

doi: 10.3892/ijo.2020.5002

Figure Lengend Snippet: 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.

Article Snippet: Anti-SNAIL (cat. no. 3879), a nt i- SLUG (c at. no. 9585), a nt i-E - c a d he r i n (cat. no. 3195), anti-N-cadherin (cat. no. 4061), anti-ERK1/2 (cat. no. 4695), anti-phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4374) and anti-GAPDH (2118L) primary antibodies were purchased from Cell Signaling Technology, Inc.; the anti-HECTD1 primary antibody (cat. no. CSB-PA010273GA01HU) was obtained from Cusabio Technology LLC; the anti-ubiquitin primary antibody (cat. no. ab7780) was obtained from Abcam; and the anti-HECTD1 (M03), clone 1E10 primary antibody (cat. no. H00025831-M03) was purchased from Abnova Corp.

Techniques: In Vivo, Expressing, Immunoprecipitation, Transfection, Western Blot, Ubiquitin Proteomics, Plasmid Preparation, Negative Control, Knockdown

Journal: International Journal of Oncology

Article Title: HECTD1 regulates the expression of SNAIL: Implications for epithelial-mesenchymal transition

doi: 10.3892/ijo.2020.5002

Figure Lengend Snippet: 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.

Article Snippet: Anti-SNAIL (cat. no. 3879), a nt i- SLUG (c at. no. 9585), a nt i-E - c a d he r i n (cat. no. 3195), anti-N-cadherin (cat. no. 4061), anti-ERK1/2 (cat. no. 4695), anti-phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4374) and anti-GAPDH (2118L) primary antibodies were purchased from Cell Signaling Technology, Inc.; the anti-HECTD1 primary antibody (cat. no. CSB-PA010273GA01HU) was obtained from Cusabio Technology LLC; the anti-ubiquitin primary antibody (cat. no. ab7780) was obtained from Abcam; and the anti-HECTD1 (M03), clone 1E10 primary antibody (cat. no. H00025831-M03) was purchased from Abnova Corp.

Techniques: Western Blot, Fluorescence, Microscopy, Transfection, Staining, Negative Control, Ubiquitin Proteomics, Knockdown

Journal: International Journal of Oncology

Article Title: HECTD1 regulates the expression of SNAIL: Implications for epithelial-mesenchymal transition

doi: 10.3892/ijo.2020.5002

Figure Lengend Snippet: HECTD1 translocates between the nucleus and cytoplasm. (A) HECTD1 shuttles between the nucleus and cytoplasm with or without EGF treatment. The subcellular localization of HECTD1 was analyzed using fluorescence microscopy. HeLa cells were treated with or without 100 ng/ml EGF for 6 min and immunostained with anti-HECTD1 antibody. The nucleus was stained with DAPI. The arrow-lines indicate the subcellular localizations of SNAIL, measured as the fluorescent intensity in individual cells. (B) The export of HECTD1 from the nucleus is sensitive to LMB treatment. HeLa cells were treated with or without 50 nM LMB for 4 h and sequentially treated with 100 ng/ml EGF. Cells were stained with anti-HECTD1 antibody and the fluorescent intensity demonstrates HECTD1 localization. Scale bar, 10 µm. (C and D) Intensity of HECTD1 nuclear signals and whole cells were analyzed and semi-quantified, and the percentage of nuclear signals are presented. Data were analyzed by the Student's t-test and presented as the means ± SD. *** P<0.001. LMB, leptomycin-B; EGF, epidermal growth factor; HECTD1, HECT domain E3 ubiquitin ligase 1. Nuclear signals of (E) HECTD1 and (F) SNAIL were analyzed in wild-type Hela cells with not treatment (NT), or with LMB (50 nM) or IVE (1 µM). The percentages of cell nuclear signal are shown. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons post hoc test. The data are shown as the means ± SD. *** P<0.001. (G) Quantification of SNAIL total signal intensities of wild-type cells were analyzed in different treatments. The data analyzed by one-way ANOVA and are shown as the means ± SD. n.s., no significant differences. More than 30 cells in each treatment condition were measured.

Article Snippet: Anti-SNAIL (cat. no. 3879), a nt i- SLUG (c at. no. 9585), a nt i-E - c a d he r i n (cat. no. 3195), anti-N-cadherin (cat. no. 4061), anti-ERK1/2 (cat. no. 4695), anti-phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4374) and anti-GAPDH (2118L) primary antibodies were purchased from Cell Signaling Technology, Inc.; the anti-HECTD1 primary antibody (cat. no. CSB-PA010273GA01HU) was obtained from Cusabio Technology LLC; the anti-ubiquitin primary antibody (cat. no. ab7780) was obtained from Abcam; and the anti-HECTD1 (M03), clone 1E10 primary antibody (cat. no. H00025831-M03) was purchased from Abnova Corp.

Techniques: Fluorescence, Microscopy, Staining, Ubiquitin Proteomics

Journal: International Journal of Oncology

Article Title: HECTD1 regulates the expression of SNAIL: Implications for epithelial-mesenchymal transition

doi: 10.3892/ijo.2020.5002

Figure Lengend Snippet: Knockdown of HECTD1 potentiates EMT. (A) Morphological changes in HeLa cells following HECTD1 knockdown with short hairpin RNA. HeLa cells transfected with Ctrl or HECTD1-KD exhibited an elongated and highly multipolar morphology. In the lower panel, cells were immunostained with an anti-α-tubulin antibody. Scale bar, 50 µm. Percentage of spindle-like phenotype (elongated) over 'cobblestone' phenotype (cubed) of cells were determined in a single experiment. (B) Wound healing assays were performed on fibronectin for 24 h in medium containing 1 µM aphidicolin and cell migration was semi-quantified. Statistical analysis was performed using the Student's t-test. * P<0.05. (C) Transwell migration assays were performed in HECTD1-KD-transfected (FI316530) Ca Ski cells. Results obtained from a single knockdown are presented. Cells in 4 randomly selected fields were counted using a microscope. Statistical analysis was performed using the Student's t-test. ** P<0.01. (D) Western blot analysis for selected EMT markers in Ctrl- or HECTD1-KD-transfected cells following EGF treatment. HeLa cells were treated with 100 ng/ml EGF for 0-120 min following overnight serum starvation. The numbers found below each lane indicate the quantified protein expression following normalization with GAPDH. Ctrl, negative control, HECTD1, HECT domain E3 ubiquitin ligase 1; KD, knockdown; EMT, epithelial-mesenchymal transition; EGF, epidermal growth factor.

Article Snippet: Anti-SNAIL (cat. no. 3879), a nt i- SLUG (c at. no. 9585), a nt i-E - c a d he r i n (cat. no. 3195), anti-N-cadherin (cat. no. 4061), anti-ERK1/2 (cat. no. 4695), anti-phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4374) and anti-GAPDH (2118L) primary antibodies were purchased from Cell Signaling Technology, Inc.; the anti-HECTD1 primary antibody (cat. no. CSB-PA010273GA01HU) was obtained from Cusabio Technology LLC; the anti-ubiquitin primary antibody (cat. no. ab7780) was obtained from Abcam; and the anti-HECTD1 (M03), clone 1E10 primary antibody (cat. no. H00025831-M03) was purchased from Abnova Corp.

Techniques: Knockdown, shRNA, Transfection, Migration, Microscopy, Western Blot, Expressing, Negative Control, Ubiquitin Proteomics

Journal: International Journal of Oncology

Article Title: HECTD1 regulates the expression of SNAIL: Implications for epithelial-mesenchymal transition

doi: 10.3892/ijo.2020.5002

Figure Lengend Snippet: Regulation of HECTD1 expression levels by miR-210 under hypoxic conditions. (A) HeLa cells were treated with 500 µM CoCl 2 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 [log 2 (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 CoCl 2 or 100 ng/ml EGF for 16 h. Expression levels were quantified by normalizing expression levels to cells not exposed to CoCl 2 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.

Article Snippet: Anti-SNAIL (cat. no. 3879), a nt i- SLUG (c at. no. 9585), a nt i-E - c a d he r i n (cat. no. 3195), anti-N-cadherin (cat. no. 4061), anti-ERK1/2 (cat. no. 4695), anti-phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4374) and anti-GAPDH (2118L) primary antibodies were purchased from Cell Signaling Technology, Inc.; the anti-HECTD1 primary antibody (cat. no. CSB-PA010273GA01HU) was obtained from Cusabio Technology LLC; the anti-ubiquitin primary antibody (cat. no. ab7780) was obtained from Abcam; and the anti-HECTD1 (M03), clone 1E10 primary antibody (cat. no. H00025831-M03) was purchased from Abnova Corp.

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Negative Control

Journal: International Journal of Oncology

Article Title: HECTD1 regulates the expression of SNAIL: Implications for epithelial-mesenchymal transition

doi: 10.3892/ijo.2020.5002

Figure Lengend Snippet: 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.

Article Snippet: Anti-SNAIL (cat. no. 3879), a nt i- SLUG (c at. no. 9585), a nt i-E - c a d he r i n (cat. no. 3195), anti-N-cadherin (cat. no. 4061), anti-ERK1/2 (cat. no. 4695), anti-phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4374) and anti-GAPDH (2118L) primary antibodies were purchased from Cell Signaling Technology, Inc.; the anti-HECTD1 primary antibody (cat. no. CSB-PA010273GA01HU) was obtained from Cusabio Technology LLC; the anti-ubiquitin primary antibody (cat. no. ab7780) was obtained from Abcam; and the anti-HECTD1 (M03), clone 1E10 primary antibody (cat. no. H00025831-M03) was purchased from Abnova Corp.

Techniques: Expressing, Immunohistochemical staining, Ubiquitin Proteomics