Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: UBA1 interacts with orthoflaviviral NS5 proteins. ( a ) UBA1 is co-immunoprecipitated with ZIKV, USUV and WNV NS5 proteins. 293 T cells were transfected with constructs expressing HA-tagged versions of each NS5 (ZIKV, USUV and WNV), EYFP (pEYFP-N1, shown as pEYFP) or an empty vector (pcDNA3.1, shown as pcDNA). NS5 proteins were immunoprecipitated with anti-HA agarose beads. ( b ) UBA1 coprecipitated with recombinant His-tagged ZIKV NS5 in assays using Ni-NTA agarose resin (Invitrogen). For the assay, 3 mg of whole-cell protein, extracted from 293 T cells, was incubated in the presence of either 90 µg of recombinant BSA or His-tagged ZIKV NS5.

Article Snippet: Primary antibodies used for WB are rabbit anti-UBA1 (Invitrogen, MA5-35786), mouse anti-HA epitope (BioLegend, reference 901501), rabbit anti-FLAG epitope (Proteintech, reference 20543–1-AP) and rabbit anti-ZIKV NS5 (GeneTex, GTX133328).

Techniques: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, Recombinant, Incubation

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: ZIKV NS5 colocalizes with UBA1 in the infected cell. Fluorescence signals emitted by UBA1 and NS5 overlap in nuclear and cytoplasmic regions of the infected cells at 36 h post-infection (merge). To detect NS5 (green) and UBA1 (red), specific rabbit anti-NS5 and mouse anti-UBA1 antibodies were used. Nuclei were stained with DAPI (blue). All signals combined are represented (merge). ( a, c ) UBA1 and NS5 are detected in the nuclei of ZIKV-infected cells. ( b, d ) Close-up view of the areas where colocalization between UBA1 and NS5 was observed. An amplification of the image selected in the dotted square area in ( a ) and (c) is provided in (b) and ( d ), respectively. ( e ) Colocalization of NS5 and UBA1 in the cytoplasmic region of A549 cells. ( f ) An amplification of the image selected in the dotted square area in ( e ) is provided. Arrows indicate those regions where colocalization is detected.

Article Snippet: Primary antibodies used for WB are rabbit anti-UBA1 (Invitrogen, MA5-35786), mouse anti-HA epitope (BioLegend, reference 901501), rabbit anti-FLAG epitope (Proteintech, reference 20543–1-AP) and rabbit anti-ZIKV NS5 (GeneTex, GTX133328).

Techniques: Infection, Fluorescence, Staining, Amplification

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: Enhanced ZIKV replication in cells overexpressing UBA1. ( a, b ) UBA1 is overexpressed in 293 T cells transfected with either pUBA1 ( a ) or pFLAG-UBA1 ( b ) when compared to untransfected cultures (mock) or cells transfected with an empty construct (pcDNA3.1, namely, pcDNA). ( a ) Larger levels of UBA1 detected in cells transfected with pUBA1; tubulin was used as a reference loading control. ( b ) Larger amounts of UBA1 detected in cells transfected with pFLAG-UBA1 when using a specific anti-UBA1 antibody. A positive band of the expected size for UBA1 was detected with an anti-FLAG antibody. Tubulin was used as a reference loading control. Representative images were included, which were obtained from two independent biological samples (two different WBs for each sample). ( c ) Virus titres obtained in cells previously transfected with pUBA1, pFLAG-UBA1 or an empty vector (pcDNA). Cells were infected with ZIKV at an m.o.i. of 1 TCID 50 /cell, and samples collected at different time points were titrated (four independent biological replicas). Significant differences with respect to mock-transfected cells (pcDNA) are indicated (two-way ANOVA, Dunnett’s multiple comparisons test; * P <0.05). ( d ) Representative WB of ZIKV NS5 and UBA1 protein levels in cells previously transfected (36 h earlier) with pUBA1, pFLAG-UBA1 or an empty vector (pcDNA); as a loading reference control, an anti-tubulin antibody was used (four independent biological replicas). ( e ) Ratio of NS5 and UBA1 protein levels relative to mock-transfected cells (empty vector) at 36 h post-infection. Tubulin was used as a reference control (four biological replicas).

Article Snippet: Primary antibodies used for WB are rabbit anti-UBA1 (Invitrogen, MA5-35786), mouse anti-HA epitope (BioLegend, reference 901501), rabbit anti-FLAG epitope (Proteintech, reference 20543–1-AP) and rabbit anti-ZIKV NS5 (GeneTex, GTX133328).

Techniques: Transfection, Construct, Control, Virus, Plasmid Preparation, Infection

Journal: American Journal of Cancer Research

Article Title: Type-2 11β-hydroxysteroid dehydrogenase promotes the metastasis of colorectal cancer via the Fgfbp1-AKT pathway

doi:

Figure Lengend Snippet: HSD11B2 up-regulates the expression of Fgfbp1 via AKT activation. A. Heat-map showing the representative 40 genes modulated by HSD11B2 overexpression in MC38 cells. B. Scatter plot showing the differentially expressed genes regulated by HSD11B2 overexpression. C. RT-PCR was used to detect the expression of the indicated genes in MC38 cells. D. HSD11B2 induced the expression of Fgfbp1 in the indicated cell lines. E. The PI3K inhibitor LY294002 or AKT silencing reduced the expression of Fgfbp1 in the indicated cell lines. F. Fgfbp1 and p-AKT expression were examined by IHC in HSD11B2-expressing metastatic lesions and VEC group. Scale bar: 50 μm. The data presented as mean ± SD from three independent experiments. NS: P≥0.05, *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The primary antibodies and their sources were as follows: HSD11B2 (cayman NO.10004549), Flag (proteintech 20543-1-AP), GAPDH (KC-5G4; KangChen Bio-tech, Inc., Shanghai, China), phospho-Akt (Ser473) (D9E), AKT (C67E7), ERK (137F5), phospho-ERK (D13.14.4E), p38 (D13E1), phospho-p38 (3D7), phospho-JNK (Thr183/Tyr185), JNK (ab110724; Epitomics), phospho-GSK-3β (Ser9) (D3A4), GSK-3β (D75D3), Fgfbp1 (Proteintech, 25006-1-AP).

Techniques: Expressing, Activation Assay, Over Expression, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Cancer Research

Article Title: Type-2 11β-hydroxysteroid dehydrogenase promotes the metastasis of colorectal cancer via the Fgfbp1-AKT pathway

doi:

Figure Lengend Snippet: Fgfbp1 promotes migration, invasion and AKT activation in CRC cells. A. Western blot was used to detect the efficiency of knockdown for Fgfbp1. B. The indicated cell lines were treated with siRNA against Fgfbp1 for 24 hours and then subjected to the CCK-8 assay. C. The indicated cell lines were treated with siRNA against Fgfbp1 for 24 hours and then subjected to the colony formation assay. D. The indicated cell lines were treated with siRNA against Fgfbp1 for 24 hours and then subjected to the transwell assay. Scale bar: 300 μm. E. Statistical comparisons of the indicated groups were performed. F. The indicated cell lines were treated with siRNA against Fgfbp1 for 48 hours and then subjected to western blot for detecting the indicated proteins. The data presented as mean ± SD from three independent experiments. NS: P≥0.05, *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The primary antibodies and their sources were as follows: HSD11B2 (cayman NO.10004549), Flag (proteintech 20543-1-AP), GAPDH (KC-5G4; KangChen Bio-tech, Inc., Shanghai, China), phospho-Akt (Ser473) (D9E), AKT (C67E7), ERK (137F5), phospho-ERK (D13.14.4E), p38 (D13E1), phospho-p38 (3D7), phospho-JNK (Thr183/Tyr185), JNK (ab110724; Epitomics), phospho-GSK-3β (Ser9) (D3A4), GSK-3β (D75D3), Fgfbp1 (Proteintech, 25006-1-AP).

Techniques: Migration, Activation Assay, Western Blot, Knockdown, CCK-8 Assay, Colony Assay, Transwell Assay

Journal: American Journal of Cancer Research

Article Title: Type-2 11β-hydroxysteroid dehydrogenase promotes the metastasis of colorectal cancer via the Fgfbp1-AKT pathway

doi:

Figure Lengend Snippet: Fgfbp1 mediates HSD11B2-induced cell migration invasion and AKT activation in CRC cells. A. CT26 and MC38 cells with HSD11B2 overexpression were treated with Fgfbp1 siRNA for 24 hours. Then, the indicated cell lines were subjected to transwell assays (the upper panel). Scale bar: 300 μm. B. Statistical comparisons of the indicated groups were performed. C. CT26 and MC38 cells with HSD11B2 overexpression were treated with Fgfbp1 siRNA for 48 hours. Western blot was performed to detect the indicated proteins. NS: P≥0.05, *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The primary antibodies and their sources were as follows: HSD11B2 (cayman NO.10004549), Flag (proteintech 20543-1-AP), GAPDH (KC-5G4; KangChen Bio-tech, Inc., Shanghai, China), phospho-Akt (Ser473) (D9E), AKT (C67E7), ERK (137F5), phospho-ERK (D13.14.4E), p38 (D13E1), phospho-p38 (3D7), phospho-JNK (Thr183/Tyr185), JNK (ab110724; Epitomics), phospho-GSK-3β (Ser9) (D3A4), GSK-3β (D75D3), Fgfbp1 (Proteintech, 25006-1-AP).

Techniques: Migration, Activation Assay, Over Expression, Western Blot

Journal: American Journal of Cancer Research

Article Title: Type-2 11β-hydroxysteroid dehydrogenase promotes the metastasis of colorectal cancer via the Fgfbp1-AKT pathway

doi:

Figure Lengend Snippet: The expression of HSD11B2 is positively correlated with Fgfbp1 and p-AKT in CRC samples. A. The protein expression of HSD11B2, Fgfbp1 and p-AKT were examined in 29 pairs of CRC tumor tissues (T) and corresponding adjacent non-tumor tissues (N) (n=29). B. Expression levels of HSD11B2, Fgfbp1 and p-AKT in CRC tumor tissues (T) and adjacent non-tumor tissues were shown. C. Correlations analysis for HSD11B2 relative to level of Fgfbp1 and p-AKT, and Fgfbp1 expression relative to level of p-AKT. NS: P≥0.05, *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The primary antibodies and their sources were as follows: HSD11B2 (cayman NO.10004549), Flag (proteintech 20543-1-AP), GAPDH (KC-5G4; KangChen Bio-tech, Inc., Shanghai, China), phospho-Akt (Ser473) (D9E), AKT (C67E7), ERK (137F5), phospho-ERK (D13.14.4E), p38 (D13E1), phospho-p38 (3D7), phospho-JNK (Thr183/Tyr185), JNK (ab110724; Epitomics), phospho-GSK-3β (Ser9) (D3A4), GSK-3β (D75D3), Fgfbp1 (Proteintech, 25006-1-AP).

Techniques: Expressing