Journal: Nature Communications

Article Title: The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

doi: 10.1038/ncomms13594

Figure Lengend Snippet: ( a ) Screening for the deubiquitinating enzymes of Nanog. HEK293T expressing the indicated proteins, the firefly luciferase activity was normalized to the renilla luciferase activity and then normalized to vector control. ( b ) HA-Nanog was co-expressed in HEK293T with the vector, Flag-USP21-LV or Flag-USP21-SV. After treating the cells with cycloheximide (CHX, 10 μg ml −1 ) for indicated time intervals, associated protein levels were analysed by western blotting (left panel). Statistical analysis of Nanog was presented on the right. Data are means±s.d. ( n =3), *** P <0.001 versus vector (Student's t -test). ( c ) Flag-Nanog was co-expressed in HEK293T with vector, HA-USP21 WT or HA-USP21-C221A. After treating cells with cycloheximide (CHX, 10 μg ml −1 ) for indicated time intervals, the protein levels were analysed by western blotting (left panel). Statistical analysis of Nanog was presented on the right. Data are means±s.d. ( n =3), * P <0.05, ** P <0.01, *** P <0.0001 versus vector (Student's t -test). ( d ) The knockdown efficiency of USP21 in E14 cells. ( e ) E14 cells were transfected with control shRNA, Usp21 shRNA3 or Usp2 shRNA and then treatment with CHX (10 μg ml −1 ). The endogenous protein levels were analysed by western blotting (top panel, the anti-USP21 antibody is from Abgent), and statistical analysis of Nanog was presented on the bottom. Data are means±s.d. ( n =3), ** P <0.01, *** P <0.001 versus shNC (Student's t -test). ( f ) E14 cells were infected with virus carrying GFP together with control shRNA or shRNA targeting USP21 or Nanog, and Nanog was detected with immunocytochemical staining on day 3. Scale bar, 50 μm. ( g , h ) Flag-USP21 and HA-Nanog were co-expressed in HEK293T cells. USP21 and Nanog were immunoprecipitated with anti-Flag ( g ) or HA ( h ) antibody, respectively, and the associated Nanog and USP21 were analysed by western blotting using either HA or Flag antibody. ( i ) GST pull-down assays indicated that USP21 interacts with Nanog directly. ( j ) Endogenous Nanog was immunoprecipitated with an antibody against Nanog from E14 cells, and the associated USP21 was detected by an anti-USP21 antibody obtained from Abgent. ( k ) Endogenous USP21 was immunoprecipitated with an antibody against USP21 (Abgent) from E14 cells. Nanog, Sox2, Oct4 and Klf4 were detected by western blotting.

Article Snippet: The rabbit polyclonal antibody against phospho-USP21 was generated using a CNDSRVPp[Ser]PVSEN peptides (Abmart, 1:500).

Techniques: Expressing, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Transfection, shRNA, Infection, Staining, Immunoprecipitation

Journal: Nature Communications

Article Title: The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

doi: 10.1038/ncomms13594

Figure Lengend Snippet: ( a ) Flag-Nanog and His-ubiquitin were co-expressed with USP21 WT or USP21-C221A in HEK293T cells. After MG132 (10 μM) treatment for 6 h, the ubiquitinated proteins were pulled down under denaturing conditions using Ni-NTA agarose beads and the ubiquitination of Nanog was detected by western blotting using an anti-Flag antibody. ( b ) His-USP21 protein was purified from E. coli , Flag-Nanog was purified by immunoprecipitated from HEK293T cells were transfected with Flag-Nanog, followed by immunoprecipitation using an anti-Flag antibody and then subjected to in vitro deubiquitination. ( c ) E14 cells stably expressing control or USP21 shRNA1 and USP21 shRNA3 were treated with MG132 (10 μM) for 6 h. Nanog was immunoprecipitated with an anti-Nanog antibody and the ubiquitination of Nanog was examined by western blotting using an anti-ubiquitin antibody. USP21 was detected by an anti-USP21 antibody from Abgent. ( d ) Alignment of the potential ubiquitin site in Nanog from different species. ( e ) WT or lysine-mutated Nanog and His-ubiquitin were co-expressed in HEK293T cells. After treatment with MG132 (10 μM) for 6 h, the ubiquitinated proteins were pulled down under denaturing conditions using Ni-NTA agarose beads and the ubiquitination of Nanog was detected by western blotting using the anti-Nanog antibody. ( f ) WT or K138/169R Nanog was expressed or co-expressed with Flag-USP21 in HEK293T cells. After treatment with CHX (10 μg ml −1 ), the protein level of Nanog was analysed by western blotting (left panel), and statistical analysis was presented on the right. Data are means±s.d. ( n =3), *** P <0.001 versus WT (Student's t -test).

Article Snippet: The rabbit polyclonal antibody against phospho-USP21 was generated using a CNDSRVPp[Ser]PVSEN peptides (Abmart, 1:500).

Techniques: Western Blot, Purification, Immunoprecipitation, Transfection, In Vitro, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

doi: 10.1038/ncomms13594

Figure Lengend Snippet: ( a ) Usp21 Floxp/Floxp ESCs were derived from Usp21 Floxp/Floxp mice and USP21 was depleted by infection with lentivirus carrying Cre recombinase. USP21 +/+ and USP21 −/− ESCs were stained with AP. Scale bar, 1 mm (for upper panel); 100 μm (for lower panel). Experiment was repeated three times. Shown are average values of triplicated results with means±s.d. ** P <0.01 versus USP21 +/+ (Student's t -test). ( b ) Morphology and AP staining of E14 cells infected with virus carrying control or specific shRNAs targeting USP21 or Nanog for 4 days. Scale bar, 100 μm. ( c ) Teratoma formation from E14 cells infected with control or USP21 shRNA. Scale bar, 100 μm. Right: haematoxylin and eosin staining showing tissues derived from three germ layers. Left: size of the teratoma. ( d , e ) AP staining (left) and statistical analysis (right) of E14 cells infected with shRNA targeting 3′UTR of USP21 or Nanog for 2 days and then rescued with shRNA-resistant USP21 for another 2 days. Scale bar, 1 mm (for upper panel); 50 μm (for lower panel). Data are means±s.d. ( n =3). *** P <0.001 versus shCon (two-way analysis of variance (ANOVA) test); ## P <0.01, ### P <0.001 versus vector (two-way ANOVA test). ( f , g ) AP staining (left) and statistical analysis (right) of E14 cells infected with USP21 shRNA for 2 days and then rescued with WT or K138/169R Nanog for another 2 days. Scale bar, 1 mm (for upper panel); 50 μm (for lower panel). Data are means±s.d. ( n =3), * P <0.05, ** P <0.01 (two-way ANOVA test), *** P <0.001 versus vector (two-way ANOVA test).

Article Snippet: The rabbit polyclonal antibody against phospho-USP21 was generated using a CNDSRVPp[Ser]PVSEN peptides (Abmart, 1:500).

Techniques: Derivative Assay, Infection, Staining, shRNA, Plasmid Preparation

Journal: Nature Communications

Article Title: The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

doi: 10.1038/ncomms13594

Figure Lengend Snippet: ( a , b ) Real-time PCR ( a ) and immunoblot ( b ) analyses of pluripotency markers and USP21 in E14 cells cultured in LIF-free condition for 5 days. Data are means±s.d. ( n =3), * P <0.05, ** P <0.01, *** P <0.001 versus day 0 (Student's t -test). The anti-USP21 antibody is from Abcam. ( c ) Upper: map of the USP21 promoter and the putative STAT3-binding sites. Bottom: ChIP–PCR analysis of E14 cultured with or without LIF for 3 days using anti-STAT3 antibody and PCR primers. IgG was used as a negative control. Enrichment of STAT3 on the USP21 promoter was calculated. Data are means±s.d. ( n =3). *** P <0.001 versus IgG (two-way analysis of variance (ANOVA) test); ## P <0.01, ### P <0.001 versus LIF(+) condition (two-way ANOVA test). ( d ) HEK293T cells were transfected with STAT3 or vector control, plus the USP21 basic promoter-Luc reporter. Protein level (top) and luciferase activity of STAT3 were measured. Data are means±s.d. ( n =3). ** P <0.01, *** P <0.001 versus STAT3 (−) condition (Student's t -test). ( e ) HEK293T cells were transfected with STAT3 and USP21 basic promoter (WT or carrying mutations in the putative STAT3-binding site) Luc reporter. Luciferase activity (top) and protein level of STAT3 (bottom) were measured. Data are means±s.d. ( n =3). ** P <0.01, *** P <0.001 (Student's t -test). ( f ) Real-time PCR and immunoblot analysis of USP21 messenger RNA (mRNA) level and protein level after STAT3 knockdown for 3 days in E14 cells. Data are means±s.d. ( n =3). * P <0.05, ** P <0.01 (Student's t -test), *** P <0.001 versus siNC (Student's t -test). USP21 were detected by an anti-USP21 antibody from Abgent. ( g ) E14 cells transfected with WT or STAT3 mutants were cultured in the presence of LIF. The mRNA and protein levels of Nanog and USP21 were analysed with PCR with reverse transcription (top) and western blotting (bottom), respectively. USP21 was detected with an anti-USP21 antibody from Abgent. Data are means±s.d. ( n =3). ** P <0.01,*** P <0.001 versus vector (Student's t -test).

Article Snippet: The rabbit polyclonal antibody against phospho-USP21 was generated using a CNDSRVPp[Ser]PVSEN peptides (Abmart, 1:500).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Binding Assay, Negative Control, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Nature Communications

Article Title: The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

doi: 10.1038/ncomms13594

Figure Lengend Snippet: ( a ) E14 cells were treated with mFGF4 (25 ng ml −1 ) in the presence of PD0325901 (1 μM) 12 h. Endogenous Nanog was immunoprecipitated with Nanog specific antibody, and the associated USP21 was examined by an anti-USP21 antibody from Abgent. ( b ) E14 cells were treated with mFGF4 (25 ng ml −1 ) in the presence of PD0325901 (1 μM) or not for 12 h. Endogenous Nanog was immunoprecipitated with an anti-Nanog-specific antibody, and polyubiquitination of Nanog was examined by western blotting with an anti-Ubiquitin antibody. USP21 was examined by an anti-USP21 antibody from Abgent. ( c ) E14 cells were treated with mFGF4 (25 ng ml −1 ) in the presence of PD0325901 (1 μM) or not for 12 h. Phos-Tag SDS–PAGE was applied to detect the band shift of USP21 caused by phosphorylation. USP21 was examined by an anti-USP21 antibody from Abgent. ( d ) E14 cells were treated with mFGF4 (25 ng ml −1 ) in the presence of PD0325901 (1 μM) or not for 12 h. Cell lysates were immunoprecipitated with antibody against ERK1/2 substrates and USP21 was detected with an anti-USP21 antibody. The anti-USP21 antibody is from Abgent. ( e ) Three conserved ERK1/2 phosphorylation motif ‘XXS/TP' (underlined) in USP21. The asterisk indicates the potential phosphorylation site of mouse USP21 at S93, 335 and 539. ( f ) HEK293T cells were transfected with ERK1, MEK1 CA and various mutants of Flag-USP21 (WT, S93A, S335A and S539A) mutants, and the Flag vector was used as control. Flag-USP21 was immunoprecipitated using the anti-Flag antibody and the phosphorylated USP21 was analysed with the anti-ERK1/2 phospho-substrate antibody. ( g ) E14 cells were treated with mFGF4 (25 ng ml −1 ) in the presence of PD0325901 (1 μM) or not for 12 h. USP21 were immunoprecipitated with an anti-USP21 antibody and then immunoblotted with an anti-phospho-USP21 antibody. The anti-USP21 antibody is from Abgent. ( h ) E14 cells stably expressing Flag-tagged WT or S539A (SA) were treated with mFGF4 (25 ng ml −1 ) in the presence of PD0325901 (1 μM) or not for 12 h. USP21 was immunoprecipitated with an anti-Flag antibody and then immunoblotted with an anti-phospho-USP21 antibody. ( i ) Phosphorylation of USP21 by ERK1 at S539 using in vitro kinase assay.

Article Snippet: The rabbit polyclonal antibody against phospho-USP21 was generated using a CNDSRVPp[Ser]PVSEN peptides (Abmart, 1:500).

Techniques: Immunoprecipitation, Western Blot, SDS Page, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Stable Transfection, Expressing, In Vitro, Kinase Assay

Journal: Nature Communications

Article Title: The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

doi: 10.1038/ncomms13594

Figure Lengend Snippet: ( a ) HA-Nanog was co-expressed with WT or S539D Flag-USP21 in HEK293T cells. The cells were treated with CHX (10 μg ml −1 ) and the protein levels of Nanog were analysed by western blotting. ( b ) HA-Nanog and His-ubiquitin were co-expressed with WT, S539D or S539A USP21 in HEK293T cells. After treatment with MG132 (10 μM) for 6 h, the ubiquitinated proteins were pulled down under denaturing conditions using Ni-NTA agarose beads and the polyubiquitination of Nanog was detected by western blotting using an anti-HA antibody. ( c ) HA-Nanog was co-expressed with WT, S539D or S539A Flag-USP21 in HEK293T cells. USP21 was immunoprecipitated with an anti-Flag antibody, and the associated Nanog was analysed by western blotting with an anti-HA antibody. ( d ) E14 cells were infected with virus containing WT, S539D or S539A Flag-USP21 for 36 h, and then treated with mFGF4 (25 ng ml −1 ) for 12 h. The protein level of Nanog was analysed by western blotting. ( e , f ) E14 cells were transfected Flag-USP21 WT/S539A/S539D. Thirty-six hours later, the transfected cells were cultured in N2B27 medium overnight and treated with mFGF4 (25 ng ml −1 ) for 12 h before alkaline phosphatase staining. Scale bar, 1 mm. AP-positive colonies were analysed. Data are means±s.d. ( n =3). * P <0.05, *** P <0.001 versus USP21 (−) (two-way analysis of variance (ANOVA) test); # P <0.05, ### P <0.001 versus N2B27 condition (two-way ANOVA test). ( g ) Haematoxylin and eosin staining of teratomas formed by E14 cells expressing WT, S539D or S539A USP21. Scale bar, 100 μm.

Article Snippet: The rabbit polyclonal antibody against phospho-USP21 was generated using a CNDSRVPp[Ser]PVSEN peptides (Abmart, 1:500).

Techniques: Western Blot, Immunoprecipitation, Infection, Transfection, Cell Culture, Staining, Expressing

Journal: Nature Communications

Article Title: The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

doi: 10.1038/ncomms13594

Figure Lengend Snippet: ( a , b ) Histone ubiquitination and methylation were analysed in E14 cells cultured in LIF-free condition ( a ) or in E14 cells stably expressing the control or shRNA targeting USP21 or Nanog ( b ). The anti-USP21 antibody is from Abcam. ( c ) Venn diagram depicting overlap between target genes of Usp21 and Nanog defined by their binding peaks (see the ‘ChIP-Seq data analysis' in the Methods section). Nanog ChIP-Seq data are referred to GEO Data Sets with project number GSE44764. ( d ) Binding of USP21 and its mutants to the Nanog-regulated genes. Data are means±s.d. ( n =3). # P <0.05, ## P <0.01, ### P <0.001 versus control (Student's t -test); * P <0.05, ** P <0.01, *** P <0.001 versus USP21 WT (Student's t -test). ( e ) The effect of USP21 and its mutants on the H2A ubiquitination at K119 at Nanog-regulated genes. Data are means±s.d. ( n =3). ## P <0.01, ### P <0.001 versus control (two-way analysis of variance (ANOVA) test); * P <0.05, ** P <0.01, *** P <0.001 versus USP21 WT (two-way ANOVA test). ( f ) USP21 affects the H3K4me3 level at Nanog-regulated genes. Data are means±s.d. ( n =3). * P <0.05, ** P <0.01 versus shCon (two-way ANOVA test). ( g ) Model showing that USP21 plays an important role in the maintenance of mESC self-renewal through regulating ubiquitination of Nanog and ubH2A.

Article Snippet: The rabbit polyclonal antibody against phospho-USP21 was generated using a CNDSRVPp[Ser]PVSEN peptides (Abmart, 1:500).

Techniques: Methylation, Cell Culture, Stable Transfection, Expressing, shRNA, Binding Assay, ChIP-sequencing