Figure 2.
Reducing DOs Impairs ESC Differentiation
(A) CCE cell proliferation at 48–96 hr after transfection with a serial dilution of Mcm5 siRNA. SC, scrambled siRNA. ∼90% transfection efficiency is achieved.
(B) Immunoblotting of total cell lysate at 72 hr after Mcm5 siRNA transfection. Fifteen picomoles Mcm5 siRNA knocked down MCM5 and had no effect on cell growth or DNA replication; hence, it was used for further analysis.
(C) TUNEL assay of ESCs after treatment with 500 μM HU or 0.075 μg/ml aphidicolin (Aph) for 48 hr. Fifteen picomoles Mcm5 or scrambled siRNA was transfected into the cells 72 hr prior to the HU and Aph treatment.
(D–K) Assays on Mcm4C/C ESCs (C1 and C2) and wild-type Mcm4+/+ ESCs (W1 and W2). (D) Cell proliferation rate analyzed over 72 hr is shown. (E) Overlay of OCT4 or SOX2 immunofluorescence images with DIC images is shown. OCT4- or SOX2-negative cells, larger than ESCs, are mostly MEF contamination in the ESC culture. (F) TUNEL assay of ESCs after treatment with HU or Aph for 48 hr is shown. (G and H) DIC images and immunofluorescence of NESTIN, respectively, of NSPCs at 96 hr after induced differentiation from ESCs are shown. (I) qRT-PCR analysis of Nestin and Sox1 expression during induced NSPC differentiation from ESCs is shown. (J) qRT-PCR data of the expression of three germ layer markers from days 1 to 13 during embryoid body (EB) differentiation from ESCs are shown. (K) DIC images of EBs at day 13 after induced differentiation from ESCs are shown.
(L) Frequency and average weight of teratomas generated from the wild-type (W1–W4) and Mcm4C/C (C1–C4) ESCs.
Error bars in (A), (D), (F), (I), and (J) all represent SEM of three independent experiments. See also Figures S1 and S2.