Fig. 3.

H19 decreases the phosphorylation level of 4E-BP1. a H19 overexpression suppresses 4E-BP1 phosphorylation in GH3 cells. At 48 h post-transfection, with an increasing dose of H19 in GH3 cells, the resulting cells were collected, and the protein levels of phosphorylated 4E-BP1 Thr70, phosphorylated 4E-BP1 Thr37/46, total 4E-BP1, total AKT, phosphorylated AKT Thr308, total S6K1 and phosphorylated S6K1 were examined by western blot. Tubulin was used as the loading control. b H19 knockdown increases the 4E-BP1 phosphorylation in GH3 cells. GH3 cells were infected with lentiviral H19 shRNA1 and shRNA-2 or a control shRNA. The protein levels of phosphorylated 4E-BP1 Thr37/46, total 4E-BP1, total AKT, phosphorylated AKT Thr308, total S6K1 and phosphorylated S6K1 were examined by western blot. c Overexpression of H19 suppresses 4E-BP1 phosphorylation in GH3 xenograft tumours. Some of the tumour tissues from Fig. 2g were homogenized in lysis buffer with a tissue homogenizer; then, the samples were centrifuged, and the supernatant was collected and used for immunoblotting analysis with the indicated antibodies. d Knockdown of H19 in GH3 xenograft tumours enhances 4E-BP1 phosphorylation. Some of the tumour tissues from Fig. 2j were homogenized in lysis buffer with a tissue homogenizer; then, the samples were centrifuged, and the supernatant was collected and used for immunoblotting analysis with the indicated antibodies. e H19 overexpression increases the amount of eIF4E complex bound to the cap analogue m7GTP Sepharose. Whole-cell lysates derived from GH3 cells that were transfected with the indicated dose of H19 were precipitated with m7GTP Sepharose beads followed by immunoblot analysis with the indicated antibodies. f H19 inhibits the total translation level in GH3 cells. ³⁵S labelling of newly synthesized protein was detected by autoradiography (left panel). Quantification of the results using ImageJ (right panel). The experiments were repeated three times. The data shown reflect relative protein synthesis normalized to the control without H19 overexpression. **p < 0.01, two-tailed Student’s t-test was used for statistical analysis