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. 2019 Feb 14;12(3):557–571. doi: 10.1016/j.stemcr.2019.01.013

Figure 2.

Figure 2

NOC Treatment Caused Rapid Apoptosis in hPSCs

(A) H9 cells were treated with NOC, FACS analysis of cell-cycle profile, and apoptosis by Hoechst 33342 and Annexin V-PI staining. The control was DMSO-treated cells (n > 3).

(B) Schematic timeline of the experiment/treatments.

(C) Cell-cycle profile and apoptosis rate of NOC-treated differentiated (RA+) H9 analyzed by Hoechst and Annexin V-PI staining and FACS (n > 3 independent experiments).

(D) Bar graph presentation of apoptosis rate in NOC-treated H9 cells (A) and differentiated H9 cells (C) Average apoptotic cell percentages are generated from three independent biological replicates (data presented as mean ± SEM; n.s., not significant; ∗∗p < 0.01, based on unpaired Student's t test).

(E) Images of H2B-mCherry cells treated with NOC; the time (in minutes) is indicated at the bottom of each frame. The arrow indicates a cell that entered prometaphase at 15 min, arrested until 255 min, and died at 280 min.

(F) Dot plot of the prometaphase duration of NOC-treated H9 cells (n = 53) and differentiated (RA+) H9 (n = 31) before cell death. Each dot represents one cell measured in the time-lapse video (data are presented as mean ± SEM; ∗∗∗∗p < 0.0001, based on unpaired Student's t test, data pooled from three independent experiments).

(G) FACS analysis of mitochondrial membrane potential by TMRE dye staining during NOC treatment. The numbers represent the percentage of cells with reduced TMRE fluorescence (n > 3 independent experiments).

(H) Western blot analysis of MCL1, BCL-XL, P-H3S10, cCaspase-3, and GAPDH protein levels (n = 3).

(I) Western blot of OCT4, BCL-XL, MCL1, BCL2, and GAPDH protein expression in undifferentiated (RA−) and differentiated (RA+) hPSCs (n > 3 independent experiments).

(J) FACS analysis of apoptosis in shNC and shTP53 H9 cells treated with NOC or etoposide by Annexin V-PI staining (n = 3 independent experiments).

(K) Western blot showing knocking down of TP53 protein by shRNA. TP53 level was significantly upregulated by etoposide treatment but not NOC treatment in shNC cells.

(L) Bar graph comparing the percentage of apoptotic cells in NOC- or etoposide-treated shNC and shTP53 H9 cells (data are presented as mean ± SEM of three independent experiments; n.s., not significant; ∗p < 0.05, ∗∗∗p < 0.001, based on unpaired Student's t test).