Figure 4.

ESCC Seed Sequence-Containing miRNAs Rescue DICER1 Knockout Defects

(A) Schematic representation of miRNA mimic transfection procedure in feeder-based condition. DOX, doxycycline.

(B) Representative AP-stained D11 conditional DICER1 knockout hESCs with or without transfection of miRNAs in feeder-based condition. In green are the six miRNAs that partially rescue the DICER1 knockout phenotype. n = 5 independent experiments. Scale bar, 5 mm.

(C) List of mature miRNAs (3p or 5p arms) transfected in D11 conditional DICER1 knockout line. In green are the mature miRNAs that significantly rescued the DICER1 knockout phenotype. The box encloses the seed sequence shared among those mature miRNAs that rescue the DICER1 knockout phenotype.

(D) Schematic representation of miRNA mimic transfection procedure in feeder-free condition.

(E) Flow cytometry of annexin-V in D11 conditional DICER1 knockout line cultured in feeder-free conditions for 8 days and transfected with mature miRNAs or control miRNA as shown in (D). For comparison between multiple experimental groups in (E) and (F), one-way ANOVA was used followed by multiple comparisons with Tukey correction. n = 3 independent experiments.

(F) qRT-PCR analysis of representative pro-apoptotic genes belonging to the extrinsic apoptotic pathway (FAS, NIK, TRAILR4) as well as intrinsic marker BIM in feeder-free condition with and without doxycycline treatment for 8 days and transfected with miR-302b, miR-372, or miR control as shown in (D). n = 3 independent experiments.

Error bars indicate SD, and significance is indicated as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; ns, not significant (p ≥ 0.05). See also Figure S4.