Fig. 5

Expression of critical dynamic factors in breast cancer cells before and after cryopreservation. a Cryopreserved and non-intervened ZR-75-1 and MDA-MB-231 cells were immunostained with anti-Vimentin and anti- Phalloidin (F-actin) antibodies. The fluorescent images demonstrated that the protein expression was significantly upregulated in the cells after cryopreservation compared to before cryopreservation. Scale bar: 50 μm. b and d The histogram represented the relative expression of Vimentin and F-actin in the breast cancer cells, respectively. Triplicate samples were included in five independent experiments (n = 5). Data were analyzed by Student’s t-test and expressed with mean ± SD. ZR-75-1 before vs. after ^**p < 0.01 for Vimentin, and ^***p < 0.001 for F-actin. MDA-MB-231 cells before vs. after ^****p < 0.0001 for Vimentin and F-actin. c Cryopreserved and non-intervened breast cancer cells were lysed and subjected to Western blotting with anti-Vimentin antibody (HSC70 as the loading control). Upper panel: Cryopreservation enhanced Vimentin expression, whereas the blot of the protein expression was undetectable in the non-intervened ZR-75-1 cells. Lower panel: The protein expression level equaled Vimentin/HSC70 ratio. Data were calculated by Student’s t-test and expressed with mean ± SD from five independent experiments (n = 5). Significantly different at ^****p < 0.0001. The Odyssey Clx (LI-COR) was applied to visualize the bands. Image J software was used to measure the band intensity. These blots were cropped. Full-length blots/gels are presented in Supplementary Figure 3