Journal: Cell Communication and Signaling : CCS

Article Title: NEK2 promotes the migration and proliferation of ESCC via stabilization of YAP1 by phosphorylation at Thr-143

doi: 10.1186/s12964-022-00898-0

Figure Lengend Snippet: NEK2 regulated expression of YAP1 and affected EMT of ESCC in vitro. A Silence NEK2 with two shRNA both decreased the expression of YAP1 and EMT biomarkers like N-cadherin and Vimentin. B Overexpression of NEK2 enhanced the migration in ESCC by Transwell assay. C Overexpression of NEK2 increased the expression of YAP1, N-Cadherin, and Vimentin. D The impairment of migration caused by Lv-shNEK2 could be partly recovered by the introduction of HA-YAP1 in Eca-109. E The reduction of N-Cadherin and Vimentin due to knockdown of NEK2 could be incompletely rescued with the elevation of YAP1 in Eca-109. All images represented one of three independently repeated experiments. ** P < 0.01, *** P < 0.001

Article Snippet: The source and application of antibody were: anti-NEK2 (sc-55601, Santa Cruz, 1:1000; abs136808, Absin, 1:1000), anti-YAP1 (sc-101199, Santa Cruz, 1:1000; GB111542, Servicebio, 1:1000; AF1615, Beyotime,1:1000), anti-GAPDH (AF0006, Beyotime, 1:1000) and anti-N-cadherin (GB111273, Servicebio, 1:1000), anti-Vimentin (GB11192, Servicebio, 1:1000), anti-Ubiquitin (AF0306, Beyotime, 1:1000), and anti-HA (AH158, Beyotime, 1:1000).

Techniques: Expressing, In Vitro, shRNA, Over Expression, Migration, Transwell Assay

Journal: Cell Communication and Signaling : CCS

Article Title: NEK2 promotes the migration and proliferation of ESCC via stabilization of YAP1 by phosphorylation at Thr-143

doi: 10.1186/s12964-022-00898-0

Figure Lengend Snippet: NEK2 influenced the oncological effect of ESCC and co-existed with YAP1 in vivo. A Knockdown of NEK2 decelerated the growth rate of Eca-109 in vivo. B Lv-NEK2 group had a compromised manifestation in average radiant efficiency and size than those in the Lv-Con group in vivo imaging. C Knockdown of NEK2 reduced the tumorigenesis of Eca-109 presented in size and weight. D The decreased NEK2 was accompanied by reduced expression and nucleus shuffling of YAP1 by IHC staining. E The downregulated expression of NEK2 simultaneously manifested with less expression and nucleus shuffling of YAP1 by IF imaging. * P < 0.05; IHC immunohistochemistry; IF immunofluorescence

Article Snippet: The source and application of antibody were: anti-NEK2 (sc-55601, Santa Cruz, 1:1000; abs136808, Absin, 1:1000), anti-YAP1 (sc-101199, Santa Cruz, 1:1000; GB111542, Servicebio, 1:1000; AF1615, Beyotime,1:1000), anti-GAPDH (AF0006, Beyotime, 1:1000) and anti-N-cadherin (GB111273, Servicebio, 1:1000), anti-Vimentin (GB11192, Servicebio, 1:1000), anti-Ubiquitin (AF0306, Beyotime, 1:1000), and anti-HA (AH158, Beyotime, 1:1000).

Techniques: In Vivo, In Vivo Imaging, Expressing, Immunohistochemistry, Imaging, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: NEK2 promotes the migration and proliferation of ESCC via stabilization of YAP1 by phosphorylation at Thr-143

doi: 10.1186/s12964-022-00898-0

Figure Lengend Snippet: NEK2 stabilized YAP1 and hindered its ubiquitination by phosphorylating it at Thr-143. A Endogenous YAP1 was subscribed to faster degradation in 12 h treatment of cycloheximide (20 μg/mL) between the Lv-Con and Lv-shNEK2 group. B The YAP1 underwent stronger ubiquitination in the Lv-shNEK2 group, which was more apparent in the presence of pretreatment MG132 (10 μM, 4 h) but still more intensified than that of the Lv-Con group. C NEK2 was pulled down by YAP1 in IP and vice versa, which was weaker in the Lv-shNEK2 group in Eca-109 in vitro. D The stability of Exogenous HA-YAP1 was also declined after knocking down on YAP1. E Sequencing map of mutation of Thr-143 and Ser-163 into Asp-143 and Asp-163, respectively. F The stability of HA-YAP1 was significantly stronger in the HA-YAP1 143D group than HA-YAP1 WT group and HA-YAP1 163D group. G The HA-YAP1 143D was less ubiquitinated than HA-YAP1 WT and HA-YAP1 163D group with pretreatment of MG132 (10 μM, 4 h). H Sequencing map of mutation of Thr-143 into Ala-143. I The HA-YAP1 was subscribed to the fastest degradation when its Thr-143 was mutated into alanine, while the lowest rate was found with the mutation to aspartic acid. J The HA-YAP1 143A group presented the most ubiquitinated, with MG132 (10 μM, 4 h) as previous, followed by the HA-YAP1 WT group and HA-YAP1 143D group. K The 3’-UTR-oriented siRNA was proved efficient to interfere with endogenous YAP but not exogenous YAP1. L The Mutation of Thr-143 into alanine on exogenous YAP1 limited the immigration of ESCC in vitro in the presence of siRNA-1 for endogenous YAP1. All experiments were repeated independently for three times. * P < 0.05, ** P < 0.01, ns not significant, IP immunoprecipitation

Article Snippet: The source and application of antibody were: anti-NEK2 (sc-55601, Santa Cruz, 1:1000; abs136808, Absin, 1:1000), anti-YAP1 (sc-101199, Santa Cruz, 1:1000; GB111542, Servicebio, 1:1000; AF1615, Beyotime,1:1000), anti-GAPDH (AF0006, Beyotime, 1:1000) and anti-N-cadherin (GB111273, Servicebio, 1:1000), anti-Vimentin (GB11192, Servicebio, 1:1000), anti-Ubiquitin (AF0306, Beyotime, 1:1000), and anti-HA (AH158, Beyotime, 1:1000).

Techniques: In Vitro, Sequencing, Mutagenesis, Immunoprecipitation