Serum starvation leads to CHEK2 phosphorylation that regulates primary cilia. A-B CHEK2 was phosphorylated in trophoblast HTR-8 cells upon serum starvation. (A) Extracts of HTR-8 cells under serum starvation (Starv.) for 0, 1, and 2 days were analyzed by Western blotting with antibodies against phosphorylated CHEK2 at Thr68 (p-CHEK2), CHEK2, and Ku70 (internal control). B Quantitative results for the relative intensity of p-CHEK2 to CHEK2 in (A). C-D Serum starvation induced primary ciliation. Quantitative results of (C) ciliated HTR-8 cells and (D) the length of primary cilia upon serum starvation for 0, 1, and 2 days. E-H (E-F) Total CHEK2 and (G-H) p-CHEK2 are localized to the base of primary cilia. CHEK2 of HTR-8 cells cultured in full-medium (FM) or serum-starved medium (Starv.) were observed by immunofluorescence staining with antibodies against CHEK2, p-CHEK2 and acetylated tubulin (Ac-tub). DNA were stained with DAPI. Scale bar: 10 μm. F and H Quantitative results for the relative intensity of CHEK2 and p-CHEK2 in (E) and (G), respectively. I-N CHEK2 inhibitor (CHEK2i) reduced CHEK2 activation upon serum starvation. I-J CHEK2i inhibited CHEK2 phosphorylation. I Extracts of HTR-8 cells in the presence or absence of CHEK2i under serum starvation were analyzed by Western blotting with antibodies against p-CHEK2, CHEK2, and HSC70. J Quantitative results for the relative intensity of p-CHEK2 to CHEK2 in I. K-N Reduction of phosphorylated CHEK2 at the ciliary base upon CHEK2i treatment. K-L Total CHEK2 and (M-N) phosphorylated p-CHEK2 of HTR-8 cells in the absence (CTL) or presence of CHEK2i under serum deprivation were observed by immunofluorescence staining with antibodies against CHEK2, p-CHEK2 and acetylated tubulin (Ac-tub). DNA were stained with DAPI. Scale bar: 10 μm. O-S Requirement of CHEK2 for primary ciliogenesis. O-P Inhibition of CHEK2 reduced primary cilia under serum starvation. O The primary cilia were reduced in CHEK2-inhibited HTR-8 cells under serum starvation. The axoneme (acetylated tubulin, Ac-tub) and ciliary membrane (ARL13b) were observed by immunofluorescence staining. DNA were stained with DAPI. Scale bars: 10 μm. P Quantitative results of ciliated HTR-8 cells. Q-S Depletion of CHEK2 reduced primary cilia under serum starvation. Q Western blot showing efficient depletion of CHEK2 by siCHEK2. R Quantitative results for the relative intensity of CHEK2 in Q. S Quantitative results of ciliated HTR-8 cells in the presence or absence of siCHEK2. **p < 0.01, ***p < 0.001, n.s.: no significance. These results are mean ± SD from three independent experiments. At least 300 cells were counted in each individual group
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