Fig 2: Numb was decreased by ZIKV infection through the ubiquitin–proteasome pathway. (A) ZIKV infection has a minimum effect on Numb mRNA level detected by RT-qPCR. The relative Numb mRNA level is shown compared with the mock-infected Vero cells. The cells were infected with the ZIKV PR strain at an MOI of 1 and harvested at 30 hpi. (B) ZIKV RNA level in the infected cells. A standard curve was included for calculating the absolute RNA level. Total ZIKV RNA is shown as copies in Log10 per well of a 24-well plate. (C) MG132 treatment restores the Numb protein levels in ZIKV-infected cells. Vero cells were infected with ZIKV at an MOI of 1 and 30 hpi and treated with MG132 or NH4Cl at a final concentration of 10 µM and 10 mM, respectively, for 6 h. The solvent DMSO was included as a control in the first two lanes. (D) ZIKV infection shortens the Numb protein half-life. The addition of cycloheximide to the Vero cells infected with ZIKV at an MOI of 10 for 24 h and treated with MG132 for 6 h. Mock-infected cells were treated similarly and included for control. (E) ZIKV infection increases the level of Numb protein polyubiquitination. ZIKV-infected Vero cells were harvested for immunoprecipitation with Numb antibody, followed by Western blotting with antibodies against ubiquitin (Ub) and Numb. Western blotting of the IP input of the cell lysate was also performed as a control. Representative data of three independent experiments are shown.

Fig 3: The expression of UBE2S, UBE2C, and Numb in different types of tumors (Oncomine database). In the diagram, the threshold parameters were set as follows: p-value of 0.01, fold change of 2, and gene rank of 10% for the cell color analysis within the cell. In each form, the number represents the number of analyses that meet the threshold across various malignancies.

Fig 4: Comparison of UBE2S, UBE2C, and Numb between ER+ and ER- breast tumors. (A) The mRNA levels in ER+ and ER- breast tumors (ER+ n = 70, ER- n = 74). ER- breast cancer shows higher levels of UBE2S and UBE2C, as well as lower Numb expression. (B) Western blot for protein levels and (C) qRT-PCR for mRNA levels in breast cell lines. One normal breast epithelial cell line (MCF10A), three ER- breast cancer cell lines (MDA-MB-231, MDA-MB-468, and SK-BR-3), and two ER+ breast cancer cell lines (MCF7 and T47D) a re adopted. Breast cancer cell lines demonstrate higher UBE2S and UBE2C and lower Numb than normal controls. ER- breast cancer cell lines show higher UBE2S and UBE2C, as well as lower Numb expression, than ER+ breast cancer cell lines. Student’s t-test was used for the statistical analysis. ***, P <0.001; ****, P <0.0001.

Fig 5: Comparison of UBE2S, UBE2C, and Numb between (A) normal breast tissues and breast tumors, as well as breast tumors of various (B) grades and (C) stages. (A) UBE2S and UBE2C are upregulated, while Numb is downregulated in breast tumors (n = 14) compared with normal breast tissues (n = 18) (Oncomine database of Ma breast, 2009). normal, normal breast tissues; cancer, breast cancer tissues). (B) With the increase in breast tumor grade (grade 1 n = 39, grade 2 n = 63, grade 3 n = 76), UBE2S and UBE2C show elevated expression while Numb exhibits decreased level. (C) With the increase in breast tumor stage (stage 1 n = 52, stage 2 n = 54, stage 3 n = 76), UBE2S and UBE2C show elevated expression while Numb expression decreases. A Student’s t-test was used for the statistical analysis. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.