Fig 2: Assessment of cell viability following siRNA transfection and exposure to freezing in MDA-MB-231 and MCF-7 breast cancer cells. (A) MDA-MB-231 and (B) MCF-7 cells. Cell viability was assayed using the Sulforhodamine B assay following transfection with siRNA and exposure to freezing and non-freezing conditions. Cells were transfected with siRNA targeting 3 aquaporins: AQP1, AQP3 and AQP5. The control group was the scrambled negative-control siRNA. *P<0.05. Si, small interfering; AQP, aquaporin.

Fig 3: Caveolin-1 (Cav1) is a crucial factor in Au-NP-mediated AQP1 expression. a bEnd.3 cells with a silenced Cav1 (bEnd.3 Cav1-KD) was established as described in Materials and Methods. The mRNA and protein expression level of Cav1 and AQP1 was obviously down-regulated. b/c Both images and quantitative data proved Au-NP-mediated FAK and AKT phosphorylation (as well as ERK de-phosphorylation) were prevented in bEnd.3 Cav1-KD (N = 6). d Au-NP-mediated AQP1 induction and (e) water permeability was also ameliorated in bEnd.3 Cav1-KD, as compared to wild-type (N = 6). These data manifested the requirement of Cav1 of bEnd.3 cell in respond to Au-NP stimulation. (* p < 0.05, ** p < 0.01, and *** p < 0.001 indicates statistically significant difference from the control group; ## p < 0.01, and ### p < 0.001 indicates statistically significant difference from the Au-NP-treated group)

Fig 4: Representative results of immunocytochemistry of aquaporin proteins (AQP1, AQP3 and AQP5) in (A) MDA-MB-231 and (B) MCF-7 cells. Upper panels show cells not exposed to freezing temperatures while lower panels show cells that were frozen at −13°C. (A) Intense staining of AQP1 and AQP3 was seen within the plasma and nuclear membranes following freezing for MDA-MB-231 cells (Fig. 3A, arrows); indicating relocation of AQP1 and AQP3 to membranes. Similar staining for AQP3 expression was observed in the plasma membrane following the freezing of MDA-MB-231 cells (Fig. 3A, arrows). AQP5 protein expression was clustered and localized in the nucleus in both treatment conditions (Fig. 3A, arrows). (B) In MCF-7 cells AQP1 and AQP3 showed localization in membranes following freezing. AQP1 had a diffused cytoplasm staining and accumulated in the plasma membrane compared with AQP1 localization in non-frozen cells (Fig. 3B, arrows); few cells had intense nuclear staining (Fig. 3B, arrows). Plasma membrane localization of AQP5 in MCF-7 cells appeared more intense following exposure to freezing temperature (Fig. 3B, arrows). There were no discernible changes in staining and localization of AQP5 in both MDA-MB-231 and MCF-7 cells in response to freezing. Scale bar, 25 µm. AQP, aquaporin.

Fig 5: Au-NPs induced aquaporin-1 (AQP1) protein expression in bEnd.3 cells. a/b The bEnd.3 cells (an immortalized mouse cerebral endothelial cell line) were exposed to Au-NPs (or Au-MPs) and the expression level of AQP1 was detected by western blots. Representative images showed an increase of AQP1 protein level in Au-NP-treated groups, whereas AQP1 protein level remained unaffected in Au-MP-treated groups. a concentration-dependent treatment; cells were incubated with 10, 50, 100 and 500 ng/mL Au-NPs for 24 h. b time-dependent treatment; cells were incubated with 500 ng/mL Au-NPs for 3, 6, 12, and 24 h. (* p < 0.05, ** p < 0.01, and *** p < 0.001 indicates statistically significant difference from the control group; N = 11). c Representative images of immunofluorescent staining, the Au-NP-induced AQP1 and the nucleus was manifested by red and blue fluorescence, respectively. A gain of red fluorescence in cell membrane and cytosol was observed in Au-NP-treated bEnd.3 cells (500 ng/mL; 24 h), as compared to control. d Transendothelial permeability assay was performed as described in Materials and Methods. Au-NP treatment (500 ng/mL; 24 h) made bEnd.3 cell more permeable to water. (* p < 0.05, indicates statistically significant difference from the control group; N = 12)