*2.1. E*ff*ect of Fractalkine on the Viability of JEG-3 Cells in Mono- and Co-Cultures*

targeted medical therapies to give reassurance to women suffering from its failure.

FKN has been demonstrated to regulate cell proliferation and inhibit apoptosis through different signalling pathways (MAPK, PI3K/AKT) in several cell types (e.g., monocytes, T cells, smooth muscle cells, glia cells, neurons) [10,46,47]. *2.1. Effect of Fractalkine on the Viability of JEG-3 Cells in Mono- and Co-Cultures*  FKN has been demonstrated to regulate cell proliferation and inhibit apoptosis through different signalling pathways (MAPK, PI3K/AKT) in several cell types (e.g., monocytes, T cells, smooth muscle

Endometrial HEC-1A cells produce FKN and trophoblast-like JEG-3 cells express fractalkine receptor, CX3CR1; therefore, we supposed that FKN might have an effect on the cell proliferation of JEG-3 cells in both mono- and co-cultures. It was revealed that viability was elevated in the monocultures of JEG-3 cells at 24 h and 48 h, but FKN did not affect the viability compared to the untreated control cells (Figure 1A), suggesting a normal cell proliferation. In case of the co-cultures, FKN influenced cell viability: at 24 h, JEG-3 cells showed significantly increased viability (Figure 1B). Comparing the viabilities of 24 h controls in mono- and co-cultures, it seems that the presence of HEC-1A cells in the co-culture affected JEG-3 proliferation, suggesting that the cell–cell interactions influenced cell cycle and viability (Figure 1A,B). These results indicate that FKN exerts different effects on mono- and co-cultures, and for the co-culture, the effect is time dependent. cells, glia cells, neurons) [10,46,47]. Endometrial HEC-1A cells produce FKN and trophoblast-like JEG-3 cells express fractalkine receptor, CX3CR1; therefore, we supposed that FKN might have an effect on the cell proliferation of JEG-3 cells in both mono- and co-cultures. It was revealed that viability was elevated in the monocultures of JEG-3 cells at 24 h and 48 h, but FKN did not affect the viability compared to the untreated control cells (Figure 1A), suggesting a normal cell proliferation. In case of the co-cultures, FKN influenced cell viability: at 24 h, JEG-3 cells showed significantly increased viability (Figure 1B). Comparing the viabilities of 24 h controls in mono- and co-cultures, it seems that the presence of HEC-1A cells in the co-culture affected JEG-3 proliferation, suggesting that the cell–cell interactions influenced cell cycle and viability (Figure 1A,B). These results indicate that FKN exerts different effects on mono- and co-cultures, and for the co-culture, the effect is time dependent.

**Figure 1.** Cell viability determinations of the mono-and co-cultured JEG-3 cells. Viability was measured using CCK-8 cell viability assay after fractalkine treatments. Viability is expressed as percentile of the total cell number of the untreated control cells. **(A)** Viability of the monocultured JEG-3 cells and **(B)** Cell viability measurements of co-cultured JEG-3 cells. Cell viability assays were made in quintuplicate in three independent experiments. The bars represent mean values and error bars represent standard deviation (SD) for three independent experiments (*n* = 3). The \* indicate *p* < 0.05 compared to the 6 h untreated control. The † indicate *p* < 0.05 compared to the 24 h untreated control. Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20. **Figure 1.** Cell viability determinations of the mono- and co-cultured JEG-3 cells. Viability was measured using CCK-8 cell viability assay after fractalkine treatments. Viability is expressed as percentile of the total cell number of the untreated control cells. (**A**) Viability of the monocultured JEG-3 cells and (**B**) Cell viability measurements of co-cultured JEG-3 cells. Cell viability assays were made in quintuplicate in three independent experiments. The bars represent mean values and error bars represent standard deviation (SD) for three independent experiments (*n* = 3). The \* indicate *p* < 0.05 compared to the 6 h untreated control. The † indicate *p* < 0.05 compared to the 24 h untreated control. Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20.

#### *2.2. Fractalkine Changes the Activation of ERK1/2, p38, JNK and AKT Signalling Pathways in Mono-and Co-Cultured JEG-3 Cells 2.2. Fractalkine Changes the Activation of ERK1*/*2, p38, JNK and AKT Signalling Pathways in Mono- and Co-Cultured JEG-3 Cells*

The increased viability of the cells suggests an enhanced proliferation that is regulated by several signalling pathways. FKN is involved in the regulation of MAPK and PI3K/AKT pathways, regulators of proliferation, differentiation and apoptosis [48,49]. We examined the phosphorylation of ERK1/2, p38, JNK (MAPKs) and AKT to reveal which pathway was affected by FKN and if there were any differences between the activation of signalling pathways with time and by increasing FKN concentrations (5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20). The increased viability of the cells suggests an enhanced proliferation that is regulated by several signalling pathways. FKN is involved in the regulation of MAPK and PI3K/AKT pathways, regulators of proliferation, differentiation and apoptosis [48,49]. We examined the phosphorylation of ERK1/2, p38, JNK (MAPKs) and AKT to reveal which pathway was affected by FKN and if there were any differences between the activation of signalling pathways with time and by increasing FKN concentrations (5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20).

In case of JEG-3 monoculture, F10 decreased ERK1/2 phosphorylation at 24 h while at 48 h, it increased it compared to the control cells (Figure 2A,C). Meanwhile, F20 treatment increased ERK1/2 In case of JEG-3 monoculture, F10 decreased ERK1/2 phosphorylation at 24 h while at 48 h, it increased it compared to the control cells (Figure 2A,C). Meanwhile, F20 treatment increased ERK1/2

phosphorylation at each time points (Figure 2A,C). F10 reduced p-p38 level significantly at 6 h and 24 h but increased again at 48 h (Figure 2A,D), while treatment with F20 caused elevation in p-p38 level at each time points (Figure 2A,D). In contrast with the aforementioned changes of MAPKs, F10 raised p-JNK level at 24 h. Treatment with F20 increased continuously the p-JNK level (Figure 2A,E). AKT showed different alterations compared to MAPKs due to FKN treatment. Phospho-AKT level was elevated by F10 at 24 h while F20 increased it only at 6 h (Figure 2A,F). The results show that the effect of fractalkine is concentration- and time-dependent; the higher FKN concentration (20 ng/mL) has a stronger and longer effect on the protein phosphorylation. ERK1/2 level at 6 h and 48 h (Figure 2B,G), while the other three pathways were triggered at 24 h and 48 h (Figure 2B,H–J). Interestingly, F20 treatment was less effective on the co-cultured JEG-3 cells. Phospho-ERK1/2 level increased at 6 h and 48 h that was similar to the F10 treatment (Figure 2B,G), although p-ERK1/2 level was reduced at 24 h. Phospho-p38 and p-JNK only elevated at 24 h (Figure 2B,H,I), and p-AKT level did not change during the whole experiment using F20 (Figure 2B,J). These result reveal that in the case of co-cultured JEG-3 cells, the strongest effect was found at 24 h and at this time point, F5 treatment was the most effective. Comparing the phosphorylation of MAPKs and AKT in mono- and co-cultures it seems that FKN exerts distinct effects on the cells

suggesting that the presence of HEC-1A cells in the co-culture modifies the action of FKN.

p-AKT levels at 24 h that correlated with the increased cell viability (Figure 2B,G–J). F10 raised p-

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 4 of 19

phosphorylation at each time points (Figure 2A,C). F10 reduced p-p38 level significantly at 6 h and 24 h but increased again at 48 h (Figure 2A,D), while treatment with F20 caused elevation in p-p38 level at each time points (Figure 2A,D). In contrast with the aforementioned changes of MAPKs, F10 raised p-JNK level at 24 h. Treatment with F20 increased continuously the p-JNK level (Figure 2A,E). AKT showed different alterations compared to MAPKs due to FKN treatment. Phospho-AKT level was elevated by F10 at 24 h while F20 increased it only at 6 h (Figure 2A,F). The results show that the effect of fractalkine is concentration- and time-dependent; the higher FKN concentration (20 ng/mL)

Regarding the co-cultures, in which the two cell types can get in contact with each other, we revealed different alterations in the protein phosphorylation patterns. Although F5 had no effect on

has a stronger and longer effect on the protein phosphorylation.

**Figure 2***.* Western blot analyses of signalling pathways regulated by fractalkine in mono- **(A)** and cocultures **(B)** JEG-3 cells. Cells were collected and pelleted after fractalkine treatments then cells were lysed and their protein contents were measured. The same amount of protein (10 µg) from each sample was separated by SDS-PAGE using 12% polyacrylamide gel, transferred by electroblotting to nitrocellulose membranes. The membranes were probed with anti-phospho-ERK1/2, anti-phosphop38, anti-phospho-JNK and anti-phospho-AKT according to the manufacturer's instruction. The experiments were repeated three times. β-actin was used as loading control. **(C**–**F)** Optical density analyses of the examined proteins in JEG-3 monocultures. **(G**–**J)** Optical density analyses of the examined proteins in co-cultured JEG-3 cells. The analyses were carried out using ImageJ software; the optical densities of the examined proteins were expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard deviation (SD) for three independent experiments (*n* = 3). The asterisks mark *p* < 0.05 compared to the appropriate controls (6 h, 24 h and 48 h). Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20. **Figure 2.** Western blot analyses of signalling pathways regulated by fractalkine in mono- (**A**) and co-cultures (**B**) JEG-3 cells. Cells were collected and pelleted after fractalkine treatments then cells were lysed and their protein contents were measured. The same amount of protein (10 µg) from each sample was separated by SDS-PAGE using 12% polyacrylamide gel, transferred by electroblotting to nitrocellulose membranes. The membranes were probed with anti-phospho-ERK1/2, anti-phospho-p38, anti-phospho-JNK and anti-phospho-AKT according to the manufacturer's instruction. The experiments were repeated three times. β-actin was used as loading control. (**C**–**F**) Optical density analyses of the examined proteins in JEG-3 monocultures. (**G**–**J**) Optical density analyses of the examined proteins in co-cultured JEG-3 cells. The analyses were carried out using ImageJ software; the optical densities of the examined proteins were expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard deviation (SD) for three independent experiments (*n* = 3). The \* mark *p* < 0.05 compared to the appropriate controls (6 h, 24 h and 48 h). Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20.

*2.3. Fractalkine Exerts Different Effects on the mRNA Expression of Proliferation, Differentiation and Invasion Regulating Genes in Mono- and Co-Cultured JEG-3 Cells*  Regarding the co-cultures, in which the two cell types can get in contact with each other, we revealed different alterations in the protein phosphorylation patterns. Although F5 had no effect on the examined signalling pathways in the monoculture, we examined the effect of F5 on the co-cultured JEG-3 cells, too. F5 did not act on the cells at 6 h and 48 h, but it elevated p-p38, p-JNK and p-AKT levels at 24 h that correlated with the increased cell viability (Figure 2B,G–J). F10 raised p-ERK1/2 level at 6 h and 48 h (Figure 2B,G), while the other three pathways were triggered at 24 h and 48 h (Figure 2B,H–J). Interestingly, F20 treatment was less effective on the co-cultured JEG-3 cells. Phospho-ERK1/2 level increased at 6 h and 48 h that was similar to the F10 treatment (Figure 2B,G), although p-ERK1/2 level was reduced at 24 h. Phospho-p38 and p-JNK only elevated at 24 h (Figure 2B,H,I), and p-AKT level did not change during the whole experiment using F20 (Figure 2B,J).

These result reveal that in the case of co-cultured JEG-3 cells, the strongest effect was found at 24 h and at this time point, F5 treatment was the most effective. Comparing the phosphorylation of MAPKs and AKT in mono- and co-cultures it seems that FKN exerts distinct effects on the cells suggesting that the presence of HEC-1A cells in the co-culture modifies the action of FKN.
