## 3 Results  
### E-Cadherin in HaCaT Cells Exposed to Simulated Microgravity  
#### 3.1.1 E-Cadherin Expression in HaCaT Exposed to Weightlessness and after Reseeding into a 1g Field  
E-cadherin expression was evaluated by Western blot analysis, as shown in Figure 1. In HaCaT cells exposed to simulated microgravity for 24 h, we observed a significant decrease in E-cadherin expression as compared to 1g controls. E-cadherin recovers normal values when the cells were placed in RPM for 48 and 72 h or when HaCaT cells were placed in RPM for 24 h and then transferred into a normal gravitational field for further 24 h (recovery). 1.2 E-Cadherin Distribution Pattern in HaCaT Exposed to Weightlessness and after Reseeding into a 1g Field  
These results are mirrored by data provided by confocal and electron microscopy studies. Indeed, as shown in Figure 2, the fluorescence intensity of E-Cadherin after 24 h in RPM-exposed cells decreases significantly, especially nearby areas of cell-to-cell contact (Figure 2B). To confirm these observations, we performed quantitative analyses of fluorescence intensity, and we found a reduction of about 30% in RPM-treated cells with respect to the 1g condition (128.354 a.u \(\pm\) 12.654 vs. 178.878 a.u \(\pm\) 10.522 respectively; \(p\) < 0.01; Table 1). Moreover, TEM analysis of HaCaT cells evidences well-organized adherens junctions in cell growing at 1g (Figure 2C), whereas a disassembled junctional complex--characterized by a reduced electron density--is noticed in RPM-treated cells (Figure 2D). \begin{table}
\begin{tabular}{c c c} \hline \hline  & **24 h 1g** & **24 h RPM** \\ \hline Fluorescence intensity ((SUM (I) a.u./cell) & 178.878 & 128.354 \\  & \(\pm\)10.522 S.E. & \(\pm\)12.654 S.E. \\ \hline Cell number & 500 & 504 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Quantitative analysis of fluorescence intensity of E-cadherin. Fluorescence intensity (SUM (I), a.u arbitrary units) was evaluated in about 500 cells. (S.E. Standard Error; 24 h 1g vs24 h RPM \(p\) < 0.01 valuated by Student’s \(t\)-test). Figure 1: Western blot analyses of E-cadherin in human keratinocytes (HaCaT) cells cultured in normal gravitational condition (1g) and microgravity conditions (random positioning machine, RPM) for 24 h, 48 h, 72 h, and after recovery experiments. Columns represent the densitometric quantification of optical density (OD) of E-cadherin signal normalized with the OD values of \(\beta\)-actin used as a loading control. Values are expressed as a fold increase of control. Values are means of three independent experiments; SD is depicted as vertical bars \({}^{**}\)\(p\) < 0.01 vs. 1g control by an unpaired two-tailed \(t\)-test. On the bottom, a panel of representative Western blot analyses is reported. For estimating the adaptive process to microgravity, HaCaT cells were placed in RPM for 48 and 72 h and then fixed and prepared for immunofluorescence experiments followed by confocal analysis. After 48 h of simulated microgravity, intense E-cadherin fluorescence is de novo appreciable within the cell-cell contact area, with only minimal differences when compared to 1g control (Figure 3A,B). At 72 h, both 1g and RPM-exposed cells show a similar distribution pattern of E-cadherin (Figure 3C,D). Overall, these findings suggest that E-cadherin recovers a normal distribution pattern after prolonged exposition to weightlessness. Similarly, cells maintained in RPM for 24 h and then transferred into a normal gravitational field for a further 24 h recover an E-cadherin distribution pattern that appears undistinguishable from that observed at 1g condition (Figure 3E,F). Moreover, TEM analysis, performed in the same experimental setting, confirms that after 48 h of microgravity exposure, adherent junctions are again observable among cells and present ultrastructural features overlapping with those noticed in cells never exposed to simulated microgravity and growing in a 1g field (Figure 3G,H).