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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: EIF3H promotes aggressiveness of esophageal squamous cell carcinoma by modulating Snail stability

Fig. 5

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

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