Fig 2: hTERT was degraded by the proteasome pathway mediated by UBE2D3(A) mRNA of hTERT in EC109-pEGFP-UBE2D3 cells was much higher than that in EC109-pEGFP cells (P = 0.000, t = 28.974) (B) Line 1 and 2 were tested before MG132 treatment, result showed that the up-regulation of UBE2D3 decreased the expression of hTERT. Line 3 and 4 were tested after 2 hours of MG132 desposed, aboundance of hTERT in EC109-pEGFP-UBE2D3 cells almost reached the same level to that in EC109-pEGFP cells. (C) hTERT protein was obtained by co-immunoprecipitation assay, and anti-ubiquitin antibody was used in immunoblotting to value the ubiquitination of hTERT. Almost discovery nothing in line 1 and 2 without MG132 treatment, but after MG132 treatment, the ubiquitin level in EC109-pEGFP-UBE2D3 cells was dramatic higher than that in EC109-pEGFP cells. Indicated that ubiquitined hTERT was up-regulated by UBE2D3 overexpressing.

Fig 3: UBE2D3 overexpression shortened telomere length and decreased telomerase activity(A) As to EC109 cells, relative telomere length of EC109-pEGFP-UBE2D3 cells was shorter than that of EC109 cells (P = 0.002, t = 5.463), while no obvious difference of relative telomere length (B) was observed between EC109-pEGFP cells and EC109 cells (P = 0.817, t = 0.253).

Fig 4: Effects of UBE2D3 overexpression on tumours in nude mice(A) EC109-pEGFP cells or EC109-pEGFP-UBE2D3 cells were subcutaneously injected into the right dorsal leg of nude mice, which were named as NC group and OE group respectively. Longest diameter “a” and the shortest diameter “b” of tumors were measured every 3 days, tumor volume (in mm3) = a × b2 × 0.5. It could be observed that UBE2D3 up-regulation could inhibit tumor growth. (B) When the volume of tumors reached 0.5 to 1.0 cm in diameter (around 20 days post injection). The mice was exposed to 10 Gy X-ray once every 6 days for a total of two exposures, it could be observed that tumors in OE group were even disappeared and smaller than that in NC group. (C) The body weight of mice was measured every 3 days, no significant difference was found between the two groups. Indicated that the effects of nutrition on tumor growth can be excluded. (D) Animals were sacrificed by cervical dislocation, before tumor tissues were excised, mice were arranged together to take photos.

Fig 5: hTERT decreased in vivo by UBE2D3 overexpressionImmunohistochemical staining of human telomerase reverse transcriptase (hTERT) protein in tumor tissues on nude mice. hTERT expression in OE group (B) was lower than that in NC group (A). Cells were enlarged 200 times by microscopy.