*2.5. Fetuin-A Inhibits Primary Cilium Formation in Placental Cells*

(microtubule marker). DNA was stained with DAPI. Scale bar 5 µM.

The primary cilium is important for maintaining placenta development. Therefore, we examined whether fetuin-A affects primary cilium formation. First, we examined whether placental HTR8 cells could grow primary cilia by forcing cells to enter the G0 phase under serum deprivation. At the G0 stage, the primary cilium, as shown by acetylated tubulin staining (axoneme marker), started to grow from the mother centriole (Cep164 staining) (Figure 6A, upper panel). Then, we examined other ciliary components. The ciliary membrane marker Arl13b and the intraflagella transporter marker IFT88

colocalized with the acetylated tubulin, suggesting that these cilia were intact. Then, we examined the effects of fetuin-A on ciliogenesis. Fetuin-A treatment inhibited primary cilia formation, as shown by the reduction of all ciliary markers (Figure 6B–E, *p* = 0.005). Thus, fetuin-A inhibits primary cilium formation in placental cells. IFT88 colocalized with the acetylated tubulin, suggesting that these cilia were intact. Then, we examined the effects of fetuin-A on ciliogenesis. Fetuin-A treatment inhibited primary cilia formation, as shown by the reduction of all ciliary markers (Figure 6B–E, *p* = 0.005). Thus, fetuin-A inhibits primary cilium formation in placental cells.

ciliary components. The ciliary membrane marker Arl13b and the intraflagella transporter marker

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 8 of 13

The primary cilium is important for maintaining placenta development. Therefore, we examined whether fetuin-A affects primary cilium formation. First, we examined whether placental HTR8 cells could grow primary cilia by forcing cells to enter the G0 phase under serum deprivation. At the G0 stage, the primary cilium, as shown by acetylated tubulin staining (axoneme marker), started to grow

*2.5. Fetuin-A Inhibits Primary Cilium Formation in Placental Cells.*

**Figure 6.** Fetuin-A inhibits ciliogenesis. (**A**–**D**) Primary cilia were examined in the absence (CTL) or presence of fetuin-A (FA) by immunostaining with antibodies against acetylated tubulin (Ace-tub), Cep164, IFT88, or Arl13b. (**E**) Quantification results of the frequency of ciliated HTR8 cells. \*\* *p* < 0.01, **Figure 6.** Fetuin-A inhibits ciliogenesis. (**A**–**D**) Primary cilia were examined in the absence (CTL) or presence of fetuin-A (FA) by immunostaining with antibodies against acetylated tubulin (Ace-tub),Cep164, IFT88, or Arl13b. (**E**) Quantification results of the frequency of ciliated HTR8 cells. \*\* *<sup>p</sup>* <sup>&</sup>lt; 0.01, results are the mean ± SD from three independent experiments, more than 100 cells were counted in each individual group. DNA was stained with DAPI. Scale bar 10 µM.
