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. 2022 Oct 10;221(12):e202107134. doi: 10.1083/jcb.202107134

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© 2022 Weier et al.

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Figure 4. — Mature DCs arrest in G1 phase. (a and b) DNA staining of mature WT and CETN2-GFP expressing BMDCs (a) and WT dermal DCs (b). Left: Representative histogram of DNA content distribution of MHCII⁺/CD11c⁺ cells. Right: Quantification of 2N and 4N cells according to DNA content. Graphs display mean values ± SD of 11/17 BMDCs (WT /CETN2-GFP) and 11 (dermal DCs) independent experiments. N = 10,000 cells (BMDCs) and at least 2,100 cells (dermal DCs) analyzed per experiment. (c) Schematic representation of nuclei labeling in FUCCI-derived BMDCs. Graphic created with BioRender.com. (d) Left: Representative images of FUCCI-derived immature and mature BMDCs. Merged and individual channels of mKO2-hCdt1 (red) and mAG-hGem (green) are shown. Scale bars, 5 μm. Right: Quantification of G1, G1–S and S/G2/M phase distribution during BMDC differentiation and maturation. Graph displays mean values ± SD of three independent experiments. At least 222 cells (immature) and 206 cells (mature) have been analyzed per condition. (e) Quantification of G1, G1–S, and S/G2/M phase distribution after FUCCI-derived BMDC stimulation with LPS. Graph displays mean values ± SD of three independent experiments. At least 228 cells per condition have been analyzed. (f) Left: Gating strategy for identification of 4N cells in G1 phase. Right: Quantification of 4N cells in G1 phase. Graph displays mean values ± SD of six independent experiments. N = 10,000 cells analyzed per experiment. (g) Quantification of centrosome numbers in immature and mature FUCCI-derived BMDCs. Left: Immunostaining of immature and mature FUCCI-derived BMDCs against ac-tubulin (white). Scale bars, 5 μm. Right: Quantification of centrosome numbers according to ac-tubulin⁺ foci. Graph displays mean values ± SD of five independent experiments. N = 246/198/237/297/200 cells (immature) and N = 298/280/291/229/190 cells (mature). ****, P < 0.0001; **, P = 0.002 (unpaired Student’s t tests corrected for multiple comparisons after Holm-Sidak). Nuclei were counterstained with DAPI. im, immature; m, mature; d, day.