Fig. 4

H19 suppresses pituitary tumour proliferation by mTORC1–4E-BP1 axis. a GH3 cells were simultaneously infected with lentiviral 4E-BP1 shRNA and 4E-BP2 shRNA or a control shRNA. 4E-BP1 and 4E-BP2 protein levels were monitored by western blot. b 4E-BP1 and 4E-BP2 double knockdown rescues H19-mediated GH3 cell growth suppression. Cell viability was detected periodically for 5 days using MTS assays and is expressed as relative proliferation (fold change over value on day 1). Error bars represent the SDs in triplicate. c A colony formation assay shows that 4E-BP1 and 4E-BP2 double knockdown rescues H19-mediated GH3 cell growth suppression. Cells were seeded into six-well plates with 200 cells per well and cultured for 10 days, followed by crystal violet staining and colony counting (scale bar, 1 cm, ***p < 0.001; n = 3). d 4E-BP1 and 4E-BP2 double knockdown rescues the growth of H19-overexpressing GH3 xenograft tumours. After mice bearing tumours were sacrificed, the tumour tissues were collected, photographed (d) and weighed (f). e The growth of GH3 xenograft tumours was measured by tumour volume. Tumour size was monitored every four days. The data (n = 5) were analysed using a sample-paired Student’s t-test; ***p < 0.001. f Tumour weight was measured in each group at the end of the experiment. Error bars are the mean ± SEM values. *p < 0.05, and **p < 0.01