Fig 1: The effect of DTX3-NOTCH2 pathway on patients with esophageal cancer. A, Representative Immunohistochemistry of NOTCH2 detected in human esophageal cancer or adjacent tissues. The tumors of patients with esophageal cancer were sliced and immunostained with anti–NOTCH2 antibody. B, The NOTCH2 density of human esophageal cancer and corresponding adjacent tissues. Every esophageal cancer sample and its corresponding adjacent tissues were connected with solid lines, **P < 0.01, very significant difference. C, Representative immunohistochemistry of DTX3 detected in human esophageal cancer. The tumors of patients with esophageal cancer were sliced and immunostained with anti–DTX3 antibody as well as H&E staining. D, The DTX3 density of human esophageal cancer and corresponding adjacent tissues. Every esophageal cancer sample and its corresponding adjacent tissues were connected with solid lines, **P < 0.01, very significant difference. E, The correlation of NOTCH2 and DTX3 expression in human esophageal cancer. **P < 0.01, very significant difference
Fig 2: Ablation of NOTCH2 inhibits the proliferation and migration of human esophageal carcinoma cells. A, The mRNA and protein profile of NOTCH2 in normal human esophageal epithelial cell line and human esophageal carcinoma cell lines detected by quantitative RT-PCR and immunoblotting. KYSE150, TE-1 and Eca-109, human esophageal carcinoma cell lines. Data expressed as mean ± SD and analyzed using one-way ANOVA with Tukey’s post-hoc test. *P < 0.05, significant difference; **P < 0.01, very significant difference; three independent experiments. B, NOTCH2 knockdown inhibits the proliferation of TE-1 and Eca-109 cells. The knockdown efficiency of shRNA for NOTCH2 was detected by immunoblotting. TE-1 and Eca-109 cells that stably expressed NOTCH2 shRNA, as well as NOTCH2 reintroduced cells, were seeded into 96-well plates and detected by CCK-8 assay at indicated time points. Six hours after cells seeded was taken as the 0-h time point. Data are expressed as mean ± SD and analyzed using two-tailed unpaired t test. *P < 0.05, significant difference; **P < 0.01, very significant difference; three independent experiments. C, NOTCH2 knockdown inhibited the colony formation of TE-1 and Eca-109 cells. TE-1 and Eca-109 cells that stably expressed Scramble or NOTCH2 shRNA, as well as NOTCH2 reintroduced cells, were seeded into 6-well-plates. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet 7 d later. The colony number was counted, with three samples in each group. Data expressed as mean ± SD and analyzed using two-tailed unpaired t test. *P < 0.05, significant difference; **P < 0.01, very significant difference; three independent experiments. D, NOTCH2 knockdown inhibited the migration of TE-1 and Eca-109 cells detected by wound-healing assay. TE-1 and Eca-109 cells that stably expressed Scramble or NOTCH2 shRNAs, as well as NOTCH2 reintroduced cells, were seeded into 6-well-plates and a wound-healing assay was done. The images were taken at 0 and 12 h, respectively (40×)
Fig 3: DTX3 promotes the degradation of NOTCH2. A, DTX3 promotes the degradation of endogenous NOTCH2. The lysates of TE-1 and Eca-109 cells that stably expressed PCDH or PCDH-DTX3 were subjected to immunoblotting analysis using anti–DTX3 or anti–NOTCH2 antibody. B, DTX3 has no obvious effect on the expression of NOTCH2 mRNA in TE-1 and Eca-109 cells. The total RNA of TE-1 and Eca-109 cells that stably expressed pCDH or pCDH-DTX3 were separated and subjected to quantitative RT-PCR analysis. Data expressed as mean ± SD and analyzed using two-tailed unpaired t test. NS, no significant difference; three independent experiments. C, DTX3 promotes the degradation of NOTCH2 in a dose-dependent manner. Eca-109 cells that were transiently transfected with different amounts of PCDH-DTX3-Flag were subjected to immunoblotting analysis using anti–NOTCH2, anti–Flag, anti–NICH or anti–GAPDH antibody. NICH, notch intracellular domain. D, DTX3-knockdown inhibits the degradation of NOTCH2, while reintroducion of DTX3 could rescue the protein levels of NOTCH2. Eca-109 cells were stably transfected with Scramble or DTX3 shRNA, and DTX3 was reintroduced in shRNA2 stably expressed cell lines. This experiment was repeated three times and the protein levels of NOTCH2 were quantitated. *P < 0.05, significant difference; **P < 0.01, very significant difference. E, Detect the effect of proteasomal inhibitor bortezomib (BTZ) and protein translation inhibitor cycloheximide (CHX) on the reduction of NOTCH2 by DTX3. Eca-109 cells transfected with pCDH or pCDH-DTX3-Flag were treated with CHX (100 µg/mL) at indicated duration or treated with BTZ (1 µmol/L) for 6 h. CHX, cycloheximide. F, The E3 ligase activity of DTX3 is required for the reduction of NOTCH2. Eca-109 cells transfected with pCDH, pCDH-DTX3-Flag or pCDH-DTX3-Flag (?RING) were treated with CHX (100 µg/mL) at indicated duration. This experiment was repeated three times and the protein levels of NOTCH2 were quantitated. *P < 0.05, significant difference; **P < 0.01, very significant difference
Fig 4: NEDD4L promoted ubiquitination and proteasomal degradation of Notch2.(A) NEDD4L was overexpressed in A549 cells, and then cell proliferation was assessed through CCK-8. (B and C) NEDD4L was overexpressed in A549 cells, and then cell growth was assessed through colony formation assay. NEDD4L was knocked down in H1299 cells, and then cell proliferation (D) and growth (E and F) was assessed using CCK-8 assay and colony formation assay, respectively. (G and H) NEDD4L-overespressed A549 cells (or A549 control cells, 1 × 107 cells in 100 µL PBS) were inoculatedsubcutaneously into BALB/c nude mice (n = 5). Tumour growth was surveyed at the indicated time-point.
Fig 5: DTX3 inhibits the proliferation of human esophageal carcinoma cells. A, DTX3 inhibits the proliferation of TE-1 and Eca-109 cells. The knockdown efficiency of shRNA forNOTCH2 was detected by immunoblotting. TE-1 and Eca-109 cells that stably expressed pCDH or pCDH-DTX3 were seeded into 96-well-plates and detected by CCK-8 assay at indicated time points. Six hours after cells seeded were taken as 0-h time point. Data expressed as mean ± SD and analyzed using two-tailed unpaired t test. *P < 0.05, significant difference; **P < 0.01, very significant difference; three independent experiments. B, DTX3 inhibits the colony formation of TE-1 and Eca-109 cells. TE-1 and Eca-109 cells that stably expressed pCDH or pCDH-DTX3 were seeded into 6-well plates; colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet 7 d later. The colony number was counted; there were three samples in each group. Data expressed as mean ± SD and analyzed using two-tailed unpaired t test. *P < 0.05, significant difference; **P < 0.01, very significant difference; three independent experiments. C, The proliferation and apoptosis-related molecular marker detected by immunoblotting. The lysates of Eca-109 cells that stably expressed Scramble or NOTCH2 shRNA1 that were transiently transfected with pCDH or pCDH-DTX3-Flag were subjected to immunoblotting analysis using indicated antibodies. Ki-67 and p-AKT, cell proliferation-related molecules; Bad and BAX, cell apoptosis-related molecules
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