Fig. 5. AQP3 regulated CD133 and pY⁷⁰⁵-STAT3 expression.

a FACS was used to test the CD133 positive cells proportion in HCCLM3 and HUH7 cell lines. Results showed AQP3 knockdown could reduce CD133+ cells proportion (**P < 0.01). b The purification of CD133+ subpopulation in HCCLM3 and HUH7 cells was performed by MACS. The sorted CD133+ cells were displayed under electron microscope and cultured for 48 h into logarithmic phase to transfected Lv-AQP3-shRNAs or Lv-AQP3-NC. c, d qRT-PCR, western blotting showed AQP3 knockdown significantly inhibited CD133 mRNA and protein expression in CD133+ HCCLM3 and HUH7 cells (**P < 0.01). e The key protein levels in JAK/STAT3 signaling pathway was tested by western blotting in HCCLM3 and HUH7 cells. Results showed AQP3 konckdown could depress the activating STAT3 (pY⁷⁰⁵-STAT3) expression. f In HCC cells, pY⁷⁰⁵-STAT3 was rarely expressed in the cytoplasm and mainly expressed in the nucleus. AQP3 konckdown could significantly decrease pY⁷⁰⁵-STAT3 nuclear translocation; g pcDNA-AQP3 was used to upregulated AQP3 expression. We found AQP3 overexpression had no influence on STAT3 expression, but could promote STAT3 activation. AG-490 treatment could inhibit STAT3 activation and eliminate the influence of AQP3 overexpression