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. Author manuscript; available in PMC: 2021 May 19.
Published in final edited form as: Cell Rep. 2021 Apr 13;35(2):108991. doi: 10.1016/j.celrep.2021.108991

Figure 3. Abnormal proliferation and altered cell-cycle distribution in FXS patient NPCs.

Figure 3.

(A) Representative images of CTR and FXS NPCs stained for Ki67, pHH3, and DAPI. Scale bar, 50 μm.

(B and C) High-content image analysis showed FXS patient NPCs have higher mitotic activity as evidenced by more pH3 and Ki67-positive cells normalized to total cells (Mann-Whitney test, **p < 0.01, ***p < 0.001, n = 4 CTR, 4 FXS NPCs, ~3,500 cells imaged per line).

(D and E) Colorimetric ELISA shows greater BrdU incorporation in FXS patient NPCs and a FXS isogenic NPC line compared with controls (Mann-Whitney test, **p < 0.01, n = 3 CTR, 3 FXS NPCs, each data point represents an average of two replicates; unpaired t test, *p < 0.05, n = 3 experiments in isogenic control-disease NPC pair. (E) Representative bivariate dot plot for DNA content (FxCycle) and DNA synthesis (EdU). Gates for cells in G0/G1, S, and G2/M phases are indicated.

(F) Cell cycle distribution of CTR and FXS NPCs after a 30-min EdU pulse demonstrates significant reduction of cells in G0/G1 and an increase of cells in the S phase. (Two-way ANOVA corrected for multiple comparisons using Holm-Sidak method, *adjusted p < 0.05, **adjusted p < 0.01, n = 3 CTR, 3 FXS NPCs.)

(G) Representative images of CTR and FXS 3D organoids stained for proliferative markers Ki67 and SOX2. Scale bar, 50 μm.

(H) Quantification of the proportion of Ki67+SOX2+ cells in CTR and FXS organoids at day 28 shows increased actively proliferating cells in FXS (ANOVA, ***p < 0.001, ANOVA, n = 3 CTR, 3 FXS).