Figure 1.
The growth of MCF-7 and MDA-MB-231 cells.
Growth curves of MCF-7 (A) and MDA-MB-231 cells (B) in control condition (ctrl) and in EW treatment. The results are the mean (SD) of six independent experiments performed in triplicate; *p<0.001 vs ctrl by Student’s t test.
Figure 2.
EW induces acini and duct-like structures development in 2D culture.
Phase contrast microscopy. (A): confluent MCF-7 cells in control condition; (B) MCF-7 treated with EW (day 7); (D): confluent MDA-MB-231 cells in control condition; (E) MDA-MB-231 cells treated with EW (day 7). EW-treated cells formed acini (*) and duct-like structures (black arrows) that branched at specific points (black arrow head) and are resting on the monolayer of confluent cells. Confocal microscopy: (C), (F), representative optical sections at central level of acini and duct like structures formed after seven days of treatment with EW. The position of cell nuclei (blue) shows the cavitation of acini and duct-like structures formed in MCF-7 cells (C) and the cavitation of duct-like and acini buds (F) formed in MDA-MB-231 cells.
Figure 3.
EW induces cell polarization in MCF-7 cells.
TEM photomicrographs of MCF-7 cells at seven days of culture. (A) Ultrastructure of an acinus formed by EW-treated MCF-7 cells (day 7). The magnification of the insert shows a luminal cell undergoing cell death. Scale bars = 1 µm (B) The left image shows the morphology of the cell in control condition; the right image shows the cells treated with EW; Bars = 1 µm. The EW-treated cells were polarized and assumed the morphology of secreting cells with the nucleus in basal position and secretory vesicles in the apical portion of cell reach in microvilli, as depicted in (C).
Figure 4.
EW induces β-casein biosynthesis.
(A) and (B): representative western blot showing the levels of the protein β-casein in MCF-7 and MDA-MB-231 cells respectively, after 7 and 11 days of treatment with EW. For each cell line is reported the densitometric quantification of β-casein, obtained normalizing the O.D. of protein bands versus the O.D. of α-tubulin bands, chosen as loading control. The results are the mean (SD) of three independent experiments; *p<0.001 vs ctrl by Student’s t test. Confocal microscopy: (C) and (D), representative optical sections of MCF-7 and MDA-MB-231 cells in control condition (ctrl) and after seven days of treatment with EW. (C) The immunostaining for β-casein (red) and cell nuclei (blue) shows the absence of β-casein in control (ctrl) cells and the presence of β-casein in the lumen of acini and duct-like structures in EW-treated MCF-7 cells. (D) Control (ctrl) cells are negative, whereas MDA-MB-231 cells treated with EW are positive for β-casein staining (red). Cell nuclei are stained in blue.
Figure 5.
Cdh1 gene expression and its regulation.
(A) Real-time PCR for Cdh1 gene in MDA-MB-231 cells treated for 5 days with EW. The results are the mean (SEM) of six independent experiments; *p<0.05 vs ctrl by Mann Whitney test. (B) Methylation of the Cdh1 gene promoter in MDA-MB-231 cells at 3 and 5 days of treatment with EW. Visible PCR product in lanes U indicates the presence of unmethylated sequences; visible PCR product in lanes M indicates the presence of methylated sequences. (C) and (D), representative western blot showing the levels of ZEB1 and ZEB2 proteins in MDA-MB-231 cells after 3 and 5 days of treatment with EW; proteins signals are shown together with α-tubulin bands, chosen as loading control. (E) Representative western blot showing the levels, after 3 and 5 days of treatment with EW, of the histone H3 trimethylated at lysine 4 (H3K4me3), and the levels of the total histone H3 (H3 tot) in MDA-MB-231 cells. Below is shown the densitometric quantification of H3K4me3, obtained normalizing the O.D. of protein bands versus the O.D. of Histone H3. (F) Representative western blot showing the levels, after 3 and 5 days of treatment with EW, of LSD1 and SNAIL proteins in MDA-MB-231 cells. Below is shown the LSD1/SNAIL ratio, obtained normalizing the O.D. of LSD1 bands versus the O.D. of SNAIL. The results are the mean (SD) of three independent experiments; *p<0.05 vs ctrl by Student’s t test.
Figure 6.
E-cadherin expression and β-catenin localization are influenced by EW.
(A) Representative western blot showing the levels of the proteins E-cadherin and β-catenin in MDA-MB-231 cells, after 7 and 11 days of treatment with EW. Alpha tubulin was used as a loading control. At the right of the panel are reported the densitometric quantification of E-cadherin and β-catenin respectively, obtained normalizing the O.D. of protein bands versus the O.D. of α-tubulin bands, chosen as loading control. The results are the mean (SD) of three independent experiments; *p<0.001 and #p<0.05 vs ctrl by Student’s t test. (B) Confocal microscopy of one representative optical section of MDA-MB-231 cells at the seventh experimental day: the immunostaining for β-catenin (green) shows a dispersed and punctuated staining in control condition (ctrl) with respect to the stronger staining that localized near the membrane perimeter for EW-treated cells. (C) Representative western blot showing the levels of the proteins CK18 and α-SMA in MDA-MB-231 cells, after 5 days of treatment with EW. Alpha tubulin was used as a loading control.
Figure 7.
Pluripotency factors expression is influenced by EW.
(A) Representative western blot showing the levels of the proteins Nanog, Klf4 and c-Myc in MDA-MB-231 cells, after 3 days of treatment with EW. Alpha tubulin was used as a loading control. (B) Densitometric quantification of Nanog, Klf4 and c-Myc respectively, obtained normalizing the O.D. of protein bands versus the O.D. of α-tubulin bands, chosen as loading control. The results are the mean (SD) of four independent experiments; *p<0.05 vs ctrl by Student’s t test.
Figure 8.
Ovalbumin is able to downregulate α-SMA biosynthesis in MDA-MB-231 cells.
Phase contrast microscopy. (A): confluent MCF-7 cells in OV condition (day 7). (B): confluent MDA-MB-231 cells in OV condition (day 7): OV-treated cells did not form acini or duct-like structures in both cell lines. (C) and (D): representative western blot showing the levels of the protein β-casein in MCF-7 and MDA-MB-231 cells respectively, after 5 days. (E) and (F): representative western blot showing the levels of the proteins E-cadherin, CK18, and α-SMA in MDA-MB-231 cells, after 5 days; α-tubulin was used as a loading control. Ctrl (control), EW (egg white), and OV (ovalbumin).