Fig. 4

EGOT regulates ITPR1 expression in cis and in trans in human cancer. a Nuclear and cytoplasmic fractions of HeLa and T47D cells were subjected to qRT-PCR. U1 is the nuclear (Nul) positive control; GAPDH is the cytoplasmic (Cyt) positive control. b RNA-FISH performed in HeLa and T47D cells. EGOT probes are red; ACTIN probes are green; and ACTIN served as the positive control (1000 × magnification). c, d Expression of pre-ITPR1 in EGOT overexpression and knockdown cells by qRT-PCR. e Combined RNase resistance and dual-RNA-FISH analysis. Before hybridization, T47D cells were treated with RNase A or RNase III. Hybridization was performed with specific probes against EGOT and ITPR1 transcripts. Nuclei are stained with DAPI. EGOT probes are red; ITPR1 probes are yellow; and ACTIN is the positive control. Arrows indicate foci (1000 × magnification). f Domain mapping of the EGOT transcript. g, h Expression of pre-ITPR1 and ITPR1 mRNA (g) and protein (h) in MCF7 cells transfected with different fragments of EGOT lentivirus. i An RNA pull-down assay was conducted using the Flag-MS2bp-MS2bs-based system followed by western blotting of lysates from MDA-MB-231, T47D, and UACC-812 cells after transfection with MS2-EGOT and MS2-vector (control). j Antibodies against hnRNPH1 were used for RIP, followed by qRT-PCR, in MCF7 and MDA-MB-231 cells. k, l ITPR1 mRNA expression was analyzed by qRT-PCR after hnRNPH1 knockdown via siRNAs (k), followed by western blotting (l). m The RNA pull-down assay was conducted using the Flag-MS2bp-MS2bs-based system in T47D cells after transfection with MS2-EGOT lentivirus along with different fragments of EGOT lentivirus, followed by western blotting. n Western blot of hnRNPH1 pulled down by F2-MS2 and mutated F2 RNA in 293 T cells. The red underlined sequences indicate the potential binding sites that were mutated into Us in F2, while the A, B and C binding sites were mutated at the same time to F2-mut ABC. Data are shown as the mean ± s.d. Student’s t-test was used for statistical analysis: * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001. Data represent at least three independent experiments