*2.1. The Expression of Fetuin-A Is Upregulated in the Placentas of Gestational Diabetes Mellitus (GDM) Patients*

Pregnant women who suffer from gestational diabetes mellitus (GDM) show higher levels of fetuin-A in their circulation [24], however, little is known about the effect of fetuin-A in the placenta. Therefore, we enrolled placental samples from twenty pregnant women with GDM and twenty non-GDM pregnant women. In the first trimester, the mean value of the body mass index (BMI) in the control group was 23.40 and that in the GDM group was 27.06. In the third trimester, the mean BMI value in the control group was 26.80 and that in the GDM group was 30.38 (Table 1). The treatment strategy for women with GDM follows the guidelines of the ADA "Management of Diabetes in Pregnancy: Standards of Medical Care in Diabetes 2019", including lifestyle management, medical nutrition therapy, and pharmacological therapy [3]. The mRNA level of fetuin-A in the placenta, which was measured by real-time PCR, was increased in patients with GDM (Figure 1A, *p* = 0.008). To further confirm this, the protein level of fetuin-A was also analyzed by immunoblotting assay. The abundance of placental fetuin-A was higher in GDM patients than in non-GDM subjects (Figure 1B,C, *p* = 0.008). Thus, the expression of fetuin-A is upregulated in the placentas of patients with GDM. Next, we tested whether the upregulation of fetuin-A in the placenta was induced by glucose. The immortalized

placental HTR8 cells were cultured with different concentrations of glucose for 72 h, and the expression of fetuin-A was examined. The abundance of fetuin-A was increased in a dose-dependent manner (Figure 1D,E, \*\* *p* = 0.007 and \*\*\* *p* = 0.0002). Thus, the expression of fetuin-A is induced by high glucose treatment in HTR8 cells.


**Table 1.** Characteristics of study population. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 3 of 13

Data are presented as mean ± SD. \* *p* < 0.05; \*\* *p* < 0.01, *t* test.

**Figure 1.** Fetuin-A is upregulated in the placentas of gestational diabetes mellitus (GDM) patients. (**A**–**C**) Fetuin-A is upregulated in the placentas of GDM patients: (**A**) quantification results of the fetuin-A mRNA level in the placentas of non-GDM and GDM women, (**B**) whole placenta extracts of non-GDM and GDM women were analyzed by immunoblot with antibodies against fetuin-A and actin, and (**C**) quantification results in (**B**). (**D**,**E**) Glucose induces the expression of fetuin-A in HTR8 **Figure 1.** Fetuin-A is upregulated in the placentas of gestational diabetes mellitus (GDM) patients. (**A**–**C**) Fetuin-A is upregulated in the placentas of GDM patients: (**A**) quantification results of the fetuin-A mRNA level in the placentas of non-GDM and GDM women, (**B**) whole placenta extracts of non-GDM and GDM women were analyzed by immunoblot with antibodies against fetuin-A and actin, and (**C**) quantification results in (**B**). (**D**,**E**) Glucose induces the expression of fetuin-A in HTR8 cells: (**D**) whole cell extracts of fetuin-A-treated HTR8 cells were analyzed by immunoblot with antibodies against fetuin-A and actin and (**E**) quantitation of the relative intensity of fetuin-A in (**E**). n.s., no significance; \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

### *2.2. Fetuin-A Inhibits Placental Cell Growth 2.2. Fetuin-A Inhibits Placental Cell Growth*

The effect of fetuin-A on placental cell growth was examined. A previous study showed that treatment with 600 µg/mL of fetuin-A for 48 h inhibited primary extravillous trophoblast cell growth [25]. Therefore, we treated HTR8 cells with 600 µg/mL of fetuin-A for 24 or 48 h, and the cell numbers were counted. At 24 h after fetuin-A treatment, the cell numbers were significantly reduced, and treatment with fetuin-A for 48 h inhibited placental cell growth to the half maximal inhibitory concentration (IC50) (Figure 2A,B, Figure 2A: *p* = 0.04 and Figure 2B: *p* = 0.0009). Thus, the following experiments were performed by treating cells with 600 µg/mL of fetuin-A for 48 h. When checking the morphology of fetuin-A-treated cells, several apoptotic bodies were observed, suggesting that fetuin-A treatment might induce apoptosis. To further confirm this, the marker of apoptosis, cleaved-caspase-3, was checked. Upon fetuin-A treatment, the level of cleaved-caspase-3 increased significantly (Figure 2C,D). Thus, fetuin-A induces apoptosis in placental cells. The effect of fetuin-A on placental cell growth was examined. A previous study showed that treatment with 600 µg/mL of fetuin-A for 48 h inhibited primary extravillous trophoblast cell growth [25]. Therefore, we treated HTR8 cells with 600 µg/mL of fetuin-A for 24 or 48 h, and the cell numbers were counted. At 24 h after fetuin-A treatment, the cell numbers were significantly reduced, and treatment with fetuin-A for 48 h inhibited placental cell growth to the half maximal inhibitory concentration (IC50) (Figure 2A,B, Figure 2A: *p* = 0.04 and Figure 2B: *p* = 0.0009). Thus, the following experiments were performed by treating cells with 600 µg/mL of fetuin-A for 48 h. When checking the morphology of fetuin-A-treated cells, several apoptotic bodies were observed, suggesting that fetuin-A treatment might induce apoptosis. To further confirm this, the marker of apoptosis, cleavedcaspase-3, was checked. Upon fetuin-A treatment, the level of cleaved-caspase-3 increased significantly (Figure 2C,D). Thus, fetuin-A induces apoptosis in placental cells.

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cells: (**D**) whole cell extracts of fetuin-A-treated HTR8 cells were analyzed by immunoblot with antibodies against fetuin-A and actin and (**E**) quantitation of the relative intensity of fetuin-A in (**E**).

**Figure 2.** Fetuin-A inhibits HTR8 cell growth. (**A**,**B**) Fetuin-A inhibits HTR8 cell growth in a timedependent manner. The cell numbers are shown as bright-field images (left panel) and quantification results (right panel) following treatment with 600 µg/mL of fetuin-A in HTR8 cells for 24 h (**A**) and 48 h (**B**). CTL: control and FA: fetuin-A. These results are the mean ± SD from three independent experiments. Scale bar 100 µM. (**C**,**D**) Fetuin-A induces apoptosis. (**C**) The apoptotic bodies (arrowhead in red) are observed upon treatment with 600 µg/mL of fetuin-A for 48 h in HTR8 cells. **Figure 2.** Fetuin-A inhibits HTR8 cell growth. (**A**,**B**) Fetuin-A inhibits HTR8 cell growth in a time-dependent manner. The cell numbers are shown as bright-field images (left panel) and quantification results (right panel) following treatment with 600 µg/mL of fetuin-A in HTR8 cells for 24 h (**A**) and 48 h (**B**). CTL: control and FA: fetuin-A. These results are the mean ± SD from three independent experiments. Scale bar 100 µM. (**C**,**D**) Fetuin-A induces apoptosis. (**C**) The apoptotic bodies (arrowhead in red) are observed upon treatment with 600 µg/mL of fetuin-A for 48 h in HTR8 cells. The mitotic cells are indicated by asterisks. The magnification is 400×. (**D**) Whole cell extracts of fetuin-A-treated HTR8 cell line were analyzed by immunoblot with antibodies against cleaved-caspase-3 (C-caspase-3) and actin. \* *p* < 0.05 and \*\* *p* < 0.01.

To further study how fetuin-A affects cell growth, the ability of cells to enter into the S phase was examined by the EdU incorporation assay. Fetuin-A treatment reduced the population of cells with EdU positive signals (Figure 3A,B, *<sup>p</sup>* <sup>=</sup> <sup>6</sup> <sup>×</sup> <sup>10</sup>−<sup>8</sup> ). Then, the S phase related cyclins, including cyclin E and cyclin A, and the activation of CDK2 were examined. The abundance of cyclin A, but not cyclin E, and the level of phosphorylated CDK2 were reduced upon fetuin-A treatment (Figure 3C–E; Figure 3D, *<sup>p</sup>* <sup>=</sup> <sup>3</sup> <sup>×</sup> <sup>10</sup>−<sup>6</sup> , and Figure 3E, *p* = 0.002). These data suggest that fetuin-A inhibits cyclin A-CDK2 activation, and thus leads to reduced S phase entry. Next, the ability of cells to enter the M phase was examined. Upon fetuin-A treatment, the mitotic index was reduced, indicating that the ability of cells to enter the M phase was reduced (Figure 4A, *p* = 0.02). In conclusion, fetuin-A inhibits placental cell growth. To further study how fetuin-A affects cell growth, the ability of cells to enter into the S phase was examined by the EdU incorporation assay. Fetuin-A treatment reduced the population of cells with EdU positive signals (Figure 3A,B, *p* = 6 × 10−8). Then, the S phase related cyclins, including cyclin E and cyclin A, and the activation of CDK2 were examined. The abundance of cyclin A, but not cyclin E, and the level of phosphorylated CDK2 were reduced upon fetuin-A treatment (Figure 3C–E; Figure 3D, *p* = 3 × 10−6, and Figure 3E, *p* = 0.002). These data suggest that fetuin-A inhibits cyclin A-CDK2 activation, and thus leads to reduced S phase entry. Next, the ability of cells to enter the M phase was examined. Upon fetuin-A treatment, the mitotic index was reduced, indicating that the ability of cells to enter the M phase was reduced (Figure 4A, *p* = 0.02). In conclusion, fetuin-A inhibits placental cell growth.

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The mitotic cells are indicated by asterisks. The magnification is 400×. (**D**) Whole cell extracts of fetuin-A-treated HTR8 cell line were analyzed by immunoblot with antibodies against cleaved-caspase-3 (C-

**Figure 3.** Fetuin-A inhibits S phase entry. (**A**,**B**) EdU incorporation was reduced in fetuin-A-treated HTR8 cells: (**A**) immunostaining of EdU (red) and DAPI (blue) in scramble control (CTL) or fetuin-A (FA) treated HTR8 cells and (**B**) quantification results of (**A**). The magnification is 200×. These results are the mean ± SD from three independent experiments and more than 1000 cells were counted in each individual group. (**C**–**E**) Fetuin-A inhibited cyclin A expression and CDK2 activation: (**C**) whole cell extracts of fetuin-A-treated HTR8 cells were analyzed by immunoblot with antibodies against cyclin A, cyclin E, CDK2, phosphorylated CDK2 at Thr160 (p-CDK2), and actin. (**D**,**E**) Quantitation of **Figure 3.** Fetuin-A inhibits S phase entry. (**A**,**B**) EdU incorporation was reduced in fetuin-A-treated HTR8 cells: (**A**) immunostaining of EdU (red) and DAPI (blue) in scramble control (CTL) or fetuin-A (FA) treated HTR8 cells and (**B**) quantification results of (**A**). The magnification is 200×. These results are the mean ± SD from three independent experiments and more than 1000 cells were counted in each individual group. (**C**–**E**) Fetuin-A inhibited cyclin A expression and CDK2 activation: (**C**) whole cell extracts of fetuin-A-treated HTR8 cells were analyzed by immunoblot with antibodies against cyclin A, cyclin E, CDK2, phosphorylated CDK2 at Thr160 (p-CDK2), and actin. (**D**,**E**) Quantitation of the relative intensity of cyclin A (**D**) and p-CDK2 in (**E**). \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

#### the relative intensity of cyclin A (**D**) and p-CDK2 in (**E**). \*\* *p* < 0.01 and \*\*\* *p* < 0.001. *2.3. Fetuin-A Induces Centrosome Amplification*

*2.3. Fetuin-A Induces Centrosome Amplification* The mitotic apparatus of the fetuin-A-treated cells was further examined. Normally, the mitotic spindle poles (γ-tubulin signals) orchestrate the mitotic spindle to align the duplicated chromosomes in the middle of the cells (Figure 4B, left panel), however, upon fetuin-A treatment, aberrant multiple The mitotic apparatus of the fetuin-A-treated cells was further examined. Normally, the mitotic spindle poles (γ-tubulin signals) orchestrate the mitotic spindle to align the duplicated chromosomes in the middle of the cells (Figure 4B, left panel), however, upon fetuin-A treatment, aberrant multiple mitotic spindle poles (cells with more than two γ-tubulin spots at M phase) were observed, accompanied by chromosome misalignment (Figure 4B, right panel and Figure 4C, *p* = 0.003). Thus, fetuin-A reduces M phase entry and disorganizes the mitotic apparatus in placental cells.

mitotic spindle poles (cells with more than two γ-tubulin spots at M phase) were observed,

**Figure 4.** Fetuin-A induces centrosome amplification. (**A**) Fetuin-A inhibited cells entering the M phase. Quantification results of the mitotic index in the absence (CTL) or presence of fetuin-A (FA). These results are the mean ± SD from three independent experiments; more than 1000 cells were counted in each individual group. (**B**,**C**) Aberrant mitotic spindle poles were induced by fetuin-A treatment: (**B**) immunofluorescence staining showed increased mitotic spindle poles (γ-tubulin staining, as shown by the arrowhead) upon fetuin-A treatment and (**C**) quantification results of (**B**). (**D**,**E**) Fetuin-A induced centrosome amplification: (**D**) immunofluorescence staining showed increased γ-tubulin numbers upon fetuin-A treatment and (**E**) quantification results of (**D**). \*\* *p* < 0.01 and \*\*\* *p* < 0.001, results are the mean ± SD from three independent experiments, more than 100 cells were counted in each individual group. Scale bar 5 µM. **Figure 4.** Fetuin-A induces centrosome amplification. (**A**) Fetuin-A inhibited cells entering the M phase. Quantification results of the mitotic index in the absence (CTL) or presence of fetuin-A (FA). These results are the mean ± SD from three independent experiments; more than 1000 cells were counted in each individual group. (**B**,**C**) Aberrant mitotic spindle poles were induced by fetuin-A treatment: (**B**) immunofluorescence staining showed increased mitotic spindle poles (γ-tubulin staining, as shown by the arrowhead) upon fetuin-A treatment and (**C**) quantification results of (**B**). (**D**,**E**) Fetuin-A induced centrosome amplification: (**D**) immunofluorescence staining showed increased γ-tubulin numbers upon fetuin-A treatment and (**E**) quantification results of (**D**). \*\* *p* < 0.01 and \*\*\* *p* < 0.001, results are the mean ± SD from three independent experiments, more than 100 cells were counted in each individual group. Scale bar 5 µM.

As centrosomes form the mitotic spindle poles for proper chromosome segregation, abnormal centrosomes might lead to the development of aberrant mitotic spindles. Thus, the centrosome numbers were counted by the staining of the marker of pericentriolar material, γ-tubulin, in fetuin-A-treated cells. When examining the centrosomal numbers, only one (before duplication) or two (after duplication) centrosomes were observed in control cells, however, the treatment of fetuin-A led to centrosome amplification, as shown by the presence of more than two γ-tubulin spots (Figure 4D,E, As centrosomes form the mitotic spindle poles for proper chromosome segregation, abnormal centrosomes might lead to the development of aberrant mitotic spindles. Thus, the centrosome numbers were counted by the staining of the marker of pericentriolar material, γ-tubulin, in fetuin-A-treated cells. When examining the centrosomal numbers, only one (before duplication) or two (after duplication) centrosomes were observed in control cells, however, the treatment of fetuin-A led to centrosome amplification, as shown by the presence of more than two γ-tubulin spots (Figure 4D,E, *p* = 0.001). Thus, fetuin-A induces centrosome amplification.
