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. 2021 Feb 19;12(2):199. doi: 10.1038/s41419-021-03487-0

Fig. 3. SNEP1-induced activation of Hh signaling and proliferation of CRC cell is mediated by SuFu degradation.

Fig. 3

A SNEP1 activates Hh signaling. HCT-116 cells with ectopic SNEP1 expression were harvested for qPCR. Data are presented as mean ± SD (n = 3). **p < 0.01. B SNEP1 facilitates CRC cell proliferation dependent on activation of the Hh Pathway. Caco2 cells were infected with LV-SNEP1 or control lentivirus, and treated with 2.5 μM GANT61. The cells were then stained with 0.5% crystal violet (w/v). The bar graph displays the mean ± SD, n = 3, **p < 0.01, N.S., not significance. C Exogenous expression of SNEP1 reduces SuFu protein levels. Protein levels of Gli2 and SuFu were detected via IB analysis of lysates isolated from lentivirus-transduced cell lines stably overexpressing SNEP1. D SNEP1 knockdown enhances SuFu protein levels. Protein levels of Gli2 and SuFu were measured by IB analysis of lysates isolated from lentivirus-transduced cells with stable SNEP1 knockdown. E MG132 rescues the reduction of SuFu protein levels by SNEP1. HEK-293T cells were transfected with a gradient Flag-SNEP1 construct for 48 h and were pretreated with the proteasome inhibitor MG132 overnight before harvesting. Lysates were examined via IB with the indicated antibodies. F SuFu degradation was attenuated by SNEP1 depletion. Cycloheximide (CHX) (100 μg/ml) was incubated for the indicated period with HEK-293T cells transfected with shRNA-SNEP1. Cell lysates were harvested for IB with anti-SuFu antibody. G Quantitative analysis of SuFu protein levels shown in F using ImageJ software. H Overexpression of SuFu decreased SNEP1-induced activation of Hh signaling. HCT-116 cells were infected with LV-SNEP1 or control lentivirus, and transfected with vector or SuFu plasmids for 48 h. qPCR were performed. Data are presented as mean ± SD (n = 3). **p < 0.01. I Endogenous complexes of SNEP1 with SuFu. Cell lysates of HT-29 cells were subjected to IP analysis with the indicated antibodies and protein-A/G beads overnight. Bound proteins were analyzed via IB. J Ectopically expressed SNEP1 interacts with SuFu. HEK-293T cells transfected with GFP-SNEP1 and Flag-SuFu plasmids were subjected to a Co-IP assay. K GST pull-down assay of SNEP1 and SuFu. The prokaryotically expressed GST-SNEP1 was incubated with cell lysates of GFP-SuFu-transfected HEK-293T cells. The protein complex pulled down by GST was detected via IB.