Fig 1: EIF3H interacts with Snail. a Vector or EIF3H-Flag plasmid was transfected in HEK293T cells for 24 h. After a 4 h treatment of 10 µM MG132, cell lysate was immunoprecipitated with anti-FLAG M2 beads, and the proteins interact with EIF3H were analyzed by mass spectrometry. Silver staining image was shown in the left panel. Representative proteins detected in mass spectrometry were shown in the right panel. b and c EIF3H-Flag was co-expressed with the Snail plasmid in HEK293T cells (b). Snail-Flag was co-transfected with the EIF3H plasmid in HEK293T cells (c). The co-IP assay was performed as described in (a). d Endogenous Snail was captured by anti-Snail antibody from KYSE510 cells, and the endogenous EIF3H and Snail were examined by immunoblotting. e Endogenous EIF3H and Snail in KYSE510 cells was detected by immunofluorescence staining. Scale bars, 30 µm. f The Flag-tagged empty vector, wild-type Snail or mutants plasmids were co-expressed with EIF3H plasmid. Extracts were subjected to the co-IP assay
Fig 2: EIF3H stabilizes and deubiquitinates Snail. a Snail was co-expressed with vector or EIF3H in HEK293T cells. The immunoblotting showed the protein level of Snail and EIF3H (left panel) and the RT-qPCR assay was performed to detect the mRNA level of Snail and EIF3H (right panel). b Snail was co-expressed with vector or EIF3H in HEK293T cells. After 24 h, cells were treated with CHX (50 µg/ml) for the indicated time intervals. Expression of Snail and EIF3H was detected (left) and the intensity of Snail expression was quantified by Image J software (right). c Snail-Flag, HA-UB plasmids with or without EIF3H were co-transfected in HEK293T cells for 24 h. After another 4 h 10 µM MG132 incubation, the ubiquitination assay was performed to detect the poly-ubiquitination of Snail. d and e The CHX pulse-chase assay was performed on EIF3H stably overexpressed in KYSE150 and KYSE510 cells. Expression of Snail was detected (left) and quantified by the Image J software (right). f and g EIF3H was knocked down by two different shRNA in KYSE150 and KYSE510 cells. Snail half-life was analyzed by CHX pulse-chase assay, by immunoblotting (left panel) and quantified (right panel). h The poly-ubiquitination of Snail in KYSE150-EIF3H (upper panel) and KYSE510-EIF3H overexpression cells (bottom panel) was assessed as described in (c). i The endogenous poly-ubiquitination level of Snail in KYSE150 (left panel) and KYSE510 EIF3H knockdown cells (right panel) was detected by the deubiquitination assay as described in (c). Results represent mean ± s.e.m. of three independent experiments. ***P < 0.001; NS: not significant
Fig 3: EIF3H and Snail levels positively correlate in ESCC. a Representative HE staining, EIF3H, Snail, E-cadherin, N-cadherin and Ki67 immunohistochemistry staining in lung tissues of shControl, shEIF3H#1 and shEIF3H#2 groups described in Fig. 3 (e). Scale bars, 100 µm. b Representative staining of EIF3H and Snail in ESCC samples. Scale bars, 100 µm. c The positive correlation was obtained in ESCC samples between EIF3H and Snail protein expression
Fig 4: Effects of EIF3H on ESCC cell motility and tumor metastasis in vitro and in vivo. EIF3H was stably overexpressed (a and b) or knocked down (c and d) in KYSE150 and KYSE510 cells, in which migration and invasion assays were performed. The migratory and invasive cells were counted. Data are obtained from three independent experiments. Scale bars, 500 µm. e EIF3H knockdown suppressed lung metastasis in vivo in mice. KYSE150 cells with or without knockdown of EIF3H were injected into the tail vein of SCID/Beige mice (n = 6) and the mice lung was harvested, weighed and stained 1 month later. HE staining was performed to analyze the nodules inside the lung. The lung weight was measured after the experiment. Representative images, representative HE staining (left panel) and quantitative results of lung weight (right panel) are shown. Scale bars, 1 mm. *P < 0.05; **P < 0.01; ***P < 0.001
Fig 5: EIF3H are required for Snail-mediated EMT in ESCC cells. a EIF3H was stably expressed in KYSE150 and KYSE510 cells. Snail, EIF3H, E-cadherin, N-cadherin and Vimentin protein levels were analyzed by Immunoblotting in KYSE150-EIF3H (left panel) and KYSE510-EIF3H overexpression cells (right panel) and quantified by the Image J software. b EIF3H was knocked down by two different shRNA in KYSE150 and KYSE510 cells. The expression of Snail, EIF3H and some EMT markers, as described in (a), were analyzed by Immunoblotting in KYSE150-shEIF3H (left panel) and KYSE510-shEIF3H cells (right panel) and quantified by the Image J software. c and d Snail was knocked down in KYSE150-EIF3H overexpression or control cells (c) or overexpressed in KYSE150-shEIF3H#1 or shControl cells (d). The Snail and EIF3H expression were analyzed by immunoblotting (left panel). The migration and invasion ability of these cells was analyzed by the transwell assay (middle panel), and the migratory and invasive cells were counted (right panel). Scale bars, 500 µm
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