Figure 2.
DICER1 Is Essential for hESC Self-Renewal
(A) Schematic representation of experimental procedure in feeder-based condition. Cells were split at 28,000 cells/cm2. DOX, doxycycline; WB, western blot; AP, alkaline phosphatase.
(B) Western blot of FLAG-DICER1∗ and total DICER1 levels of three conditional DICER1 knockout lines (B2, D11, F2) in feeder-based condition with and without DOX treatment for 6 days. WT, wild-type.
(C) qRT-PCR analysis of DICER1∗ transgene expression in three conditional DICER1 knockout lines in feeder-based condition with and without doxycycline treatment for 6 days. Levels relative to GAPDH. Statistical analysis was performed in comparison with +DOX samples. n = 3 independent experiments. n.d., not detected.
(D) Mature miRNA qRT-PCR analysis of eight hESC-expressed miRNAs in DICER1 conditional knockout line B2 after 6 days of DOX withdrawal in feeder-based condition. Levels are normalized to +DOX. Statistical analysis was performed in comparison with +DOX samples. n = 3 independent experiments.
(E) qRT-PCR analysis of four hESC-abundant miRNAs (miR-373, miR-302a, miR-92a, and miR-200c) at days 6 through 9 of DOX withdrawal. miRNA levels in the +DOX samples are used for comparison. Analysis was done on the B2 DICER1 conditional knockout line in feeder-based condition. For each time point, statistical analysis was performed in comparison with +DOX samples. Day-6 data are reused from (D) for comparison purposes. n = 3 independent experiments.
(F) AP staining of three conditional DICER1 knockout lines in feeder-based conditions with and without DOX treatment for 12 days. n = 6 independent experiments. Scale bar, 5 mm.
(G) AP staining of three conditional DICER1 knockout lines in feeder-free condition with and without doxycycline treatment for 8 days. n = 3 independent experiments. Scale bar, 5 mm.
Error bars indicate SD, and significance is indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant (p ≥ 0.05). See also Figure S2.