Figure 1.

Mettl8 Is Transcriptionally Regulated by STAT3

(A) Real-time PCR was performed to screen for changes when ESCs were treated with STA-21 and STATTIC for 1 hr.

(B and C) E14 cells were treated with STA-21 and STATTIC for 6 hr and harvested. (B) Total RNAs were extracted and followed by real-time PCR analysis. Data are shown as the mean ± SD from three independent experiments. ^∗p < 0.05. (C) Cell lysates were analyzed by western blot. The value of each band was calculated from three independent replicates and indicates the relative expression level after normalizing to the loading control actin.

(D) Knockdown Stat3 in E14 cells resulted in downregulation of Mettl8 mRNA. Data are shown as the mean ± SD from three independent experiments.

(E) Knockdown Stat3 in E14 cells resulted in downregulation of METTL8 protein. The value of each band was calculated from three independent replicates and indicates the relative expression level after normalizing to the loading control actin.

(F and G) E14 cells were transfected with Flag-vector or Flag-tagged STAT3 at increasing concentrations. (F) Total RNAs were extracted followed by real-time PCR analysis. Data are shown as the mean ± SD from three independent experiments. ^∗p < 0.05. (G) Cell lysates were analyzed by western blot.

(H) Bioinformatic analysis identifies three possible STAT3 binding sites on Mettl8 gene labeled as P1, P2, and P3. Data are shown as the mean ± SD from three independent experiments.

(I) Inducible Flag-METTL8 overexpression E14 cells were treated with or without doxycycline and culture in mediums with or without LIF for 6 days. Then colonies were stained for AP activity and cell lysates were analyzed by western blot. Scale bars, 0.5 cm.