Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Clinical Significance of Ubiquitin Specific Protease 7 (USP7) in Predicting Prognosis of Hepatocellular Carcinoma and its Functional Mechanisms

doi: 10.12659/MSM.909368

Figure Lengend Snippet: Clinical characteristics of enrolled HCC patients.

Article Snippet: The USP7 DNA construct was purchased from Addgene ( https://www.addgene.org/16655/ ).

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Clinical Significance of Ubiquitin Specific Protease 7 (USP7) in Predicting Prognosis of Hepatocellular Carcinoma and its Functional Mechanisms

doi: 10.12659/MSM.909368

Figure Lengend Snippet: USP7 is upregulated in HCC tumor tissues. ( A ) RT-PCR results showed that the RNA level of USP7 was increased in HCC tumor tissues than that in adjacent normal liver tissues, and was positively correlated with the TNM stage. ( B ) Representative IHC result showed the negative protein expression of USP7 in normal liver tissues. ( C ) Representative IHC result showed the high protein level of USP7 in HCC tissues, which predominately located in the nucleus. ( D ) Statistical analysis of IHC scores in normal liver tissues and HCC tissues showed a significantly higher USP7 protein level in tumor tissues. Magnification: ×400. Data was presented as mean ±SD. * P<0.05 by Student’s t-test.

Article Snippet: The USP7 DNA construct was purchased from Addgene ( https://www.addgene.org/16655/ ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Clinical Significance of Ubiquitin Specific Protease 7 (USP7) in Predicting Prognosis of Hepatocellular Carcinoma and its Functional Mechanisms

doi: 10.12659/MSM.909368

Figure Lengend Snippet: Kaplan-Meir survival curves of HCC patients. Overall survival analyses were conducted by Kaplan-Meir method and compared by log-rank test according to patients’ age ( A ), gender ( B ), tumor number ( C ), tumor size ( D ), TNM stage ( E ), and USP7 protein level ( F ). * P<0.05 by log-rank test.

Article Snippet: The USP7 DNA construct was purchased from Addgene ( https://www.addgene.org/16655/ ).

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Clinical Significance of Ubiquitin Specific Protease 7 (USP7) in Predicting Prognosis of Hepatocellular Carcinoma and its Functional Mechanisms

doi: 10.12659/MSM.909368

Figure Lengend Snippet: Kaplan-Meier overall survival of enrolled HCC patients.

Article Snippet: The USP7 DNA construct was purchased from Addgene ( https://www.addgene.org/16655/ ).

Techniques: Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Clinical Significance of Ubiquitin Specific Protease 7 (USP7) in Predicting Prognosis of Hepatocellular Carcinoma and its Functional Mechanisms

doi: 10.12659/MSM.909368

Figure Lengend Snippet: Cox multivariate analysis of enrolled HCC patients.

Article Snippet: The USP7 DNA construct was purchased from Addgene ( https://www.addgene.org/16655/ ).

Techniques: Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Clinical Significance of Ubiquitin Specific Protease 7 (USP7) in Predicting Prognosis of Hepatocellular Carcinoma and its Functional Mechanisms

doi: 10.12659/MSM.909368

Figure Lengend Snippet: USP7 enhances tumor cell proliferation, migration, and invasion. The proliferation curves of HCC cells were plotted by MTT assays, which showed that USP7 can enhance the cell viability in both Hep3B ( A ) and Huh7 ( B ) cells. Wound-healing assays were performed to evaluate the migration capacity of HCC cells, indicating a promoting role of USP7 on cell migration ( C, D ). The invasion process of tumor cells was monitored by Matrigel-transwell assay, demonstrating that USP7-overexpression enhanced cell invasion, while USP7-knockdown suppressed tumor invasion ( E, F ). Data was presented as mean ±SD. * P<0.05 by Student’s t-test.

Article Snippet: The USP7 DNA construct was purchased from Addgene ( https://www.addgene.org/16655/ ).

Techniques: Migration, Transwell Assay, Over Expression, Knockdown

Journal: European journal of histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma.

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: Figure 3. USP7 promotes FGFR1 deubiquitination to stabilize FGFR1 protein. A) Ubibrowser 2.0 database predicted the USP7-FGFR1 interaction. B) Immunoblots of si-USP7-transfected or si-NC-introduced TPC-1 and SW579 cells for evaluation of USP7 protein expres- sion. C,D) FGFR1 mRNA expression analysis by RT-qPCR (C) and FGFR1 protein level analysis by immunoblot assay (D) in si-USP7- transfected or si-NC-introduced TPC-1 and SW579 cells. E) Representative immunoblotting revealing FGFR1 protein level in TPC-1 and SW579 cells after si-NC introduction, si-USP7 transfection, or si-USP7 transfection before MG132 exposure (100 nM, 24 h). F) TPC-1 cells were co-transfected with Flag-FGFR1 plasmid and si-USP7 or sh-NC control, followed by immunoprecipitation (IP) experiment with anti-Flag antibody; the immunoprecipitates were subjected to immunoblot assay (IB) using anti-Ub antibody. G) Representative immuno- fluorescence staining depicting the expression and localization of USP7 and FGFR1 in si-USP7-transfected or si-NC-introduced TPC-1 cells. *p<0.05.

Article Snippet: We determined the influence of USP7 in FGFR1 deubiquitination by an immunoprecipitation experiment with recombinant antiFlag antibody (#80010-1-RR, 1 μg for 1 mg of protein extract; Proteintech) and subsequent immunoblot analysis using rabbit ubiquitin polyclonal antibody (anti-Ub, #10201-2-AP, 1:5000; Proteintech).

Techniques: Western Blot, Transfection, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Immunoprecipitation, Fluorescence, Staining

Journal: European journal of histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma.

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: Figure 4. USP7 correlates with PTC cell malignant behaviors by stabilizing FGFR1. A) Immunoblots of lysates of TPC-1 and SW579 PTC cells that were introduced with si-USP7, si-USP7+FGFR1 plasmid or scrambled siRNA sequence (si-NC) for evaluation of FGFR1 protein. B) Viability analysis of TPC-1 and SW579 PTC cells transfected as in A by CCK-8 assay. C,D) Proliferative ability evaluation of TPC-1 and SW579 PTC cells transfected as in A by EdU assay. E) Apoptosis analysis of TPC-1 and SW579 PTC cells treated as in A by flow cytometry based Annexin V-FITC and PI staining. F,G) Assessment of invasiveness of TPC-1 and SW579 PTC cells introduced as in A by transwell assay. H) Determination of migratory ability of TPC-1 and SW579 PTC cells introduced as in A by would healing assay. *p<0.05.

Article Snippet: We determined the influence of USP7 in FGFR1 deubiquitination by an immunoprecipitation experiment with recombinant antiFlag antibody (#80010-1-RR, 1 μg for 1 mg of protein extract; Proteintech) and subsequent immunoblot analysis using rabbit ubiquitin polyclonal antibody (anti-Ub, #10201-2-AP, 1:5000; Proteintech).

Techniques: Western Blot, Plasmid Preparation, Sequencing, Transfection, CCK-8 Assay, EdU Assay, Flow Cytometry, Staining, Transwell Assay

Journal: European journal of histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma.

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: Figure 5. TF YY1 can promote FGFR1 transcription. A) Schematic of the motif of YY1, the predicted pairing sequence for YY1 in the FGFR1 promoter, the mutation in the pairing sequence. B) ChIP experiments of chromatin of TPC-1 and SW579 cells using antibodies against YY1 and IgG, followed by DNA purification and enrichment analysis of the FGFR1 promoter. C) Immunoblots of lysates of si- YY1- or si-NC-transfected TPC-1 and SW579 cells to assess YY1 protein expression. D,E) The reporter construct WT-FGFR1 or MUT- FGFR1 was introduced into TPC-1 and SW579 cells along with si-YY1 or si-NC control, followed by analysis of luciferase activity. F) Representative immunoblotting showing FGFR1 protein level in si-YY1- or si-NC-introduced TPC-1 and SW579 cells. *p<0.05.

Article Snippet: We determined the influence of USP7 in FGFR1 deubiquitination by an immunoprecipitation experiment with recombinant antiFlag antibody (#80010-1-RR, 1 μg for 1 mg of protein extract; Proteintech) and subsequent immunoblot analysis using rabbit ubiquitin polyclonal antibody (anti-Ub, #10201-2-AP, 1:5000; Proteintech).

Techniques: Sequencing, Mutagenesis, DNA Purification, Western Blot, Transfection, Expressing, Construct, Control, Luciferase, Activity Assay

Journal: European Journal of Histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: Enhanced expression of FGFR1 in human PTC. A ) Representative immunohistochemical staining of PTC tumors (n=3) and neighboring healthy thyroid samples (n=3). B,C ) Quantitative PCR by RT-qPCR of FGFR1 mRNA ( B ) and immunoblots of FGFR1 protein expression ( C ) in PTC tumors (n=32) and neighboring healthy thyroid samples (n=32). D) Representative immunoblot analysis of three PTC cell lines (IHH-4, TPC-1, and SW579) and control cell line Nthy-ori3-1 to detect FGFR1 protein levels. * p <0.05.

Article Snippet: The primary antibodies, including rabbit anti-FGFR1 polyclonal (#GB115541, 1:100; Servicebio) and mouse anti-USP7 monoclonal (#66514-1-Ig, 1:300; Proteintech), were used prior to the application of Alexa 488-labeled anti-mouse (#GB25301, 1:500; Servicebio) and Cy3-labeled anti-rabbit (#GB21303, 1:500; Servicebio) IgG secondary antibodies following the vendors’ guidelines.

Techniques: Expressing, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control

Journal: European Journal of Histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: Regulatory functions of FGFR1 in PTC cell phenotypes. A ) Representative immunoblot showing the protein expression of FGFR1 in TPC-1 and SW579 PTC cells that were introduced with a FGFR1-siRNA (si-FGFR1) or scrambled siRNA sequence (si-NC). B ) CCK-8 cell viability analysis of TPC-1 and SW579 PTC cells transfected as in A. C ) EdU cell proliferation assay of TPC-1 and SW579 PTC cells transfected as in A. D ) Annexin V-FITC and PI staining-based cell apoptosis analysis of TPC-1 and SW579 PTC cells treated as in A. E,F ) Transwell invasiveness analysis of TPC-1 and SW579 PTC cells introduced as in A. G ) Would healing assay of TPC-1 and SW579 PTC cells introduced as in A to test cell migratory ability. * p <0.05.

Article Snippet: The primary antibodies, including rabbit anti-FGFR1 polyclonal (#GB115541, 1:100; Servicebio) and mouse anti-USP7 monoclonal (#66514-1-Ig, 1:300; Proteintech), were used prior to the application of Alexa 488-labeled anti-mouse (#GB25301, 1:500; Servicebio) and Cy3-labeled anti-rabbit (#GB21303, 1:500; Servicebio) IgG secondary antibodies following the vendors’ guidelines.

Techniques: Western Blot, Expressing, Sequencing, CCK-8 Assay, Transfection, Proliferation Assay, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: USP7 promotes FGFR1 deubiquitination to stabilize FGFR1 protein. A ) Ubibrowser 2.0 database predicted the USP7-FGFR1 interaction. B ) Immunoblots of si-USP7-transfected or si-NC-introduced TPC-1 and SW579 cells for evaluation of USP7 protein expression. C,D ) FGFR1 mRNA expression analysis by RT-qPCR ( C ) and FGFR1 protein level analysis by immunoblot assay ( D ) in si-USP7-transfected or si-NC-introduced TPC-1 and SW579 cells. E ) Representative immunoblotting revealing FGFR1 protein level in TPC-1 and SW579 cells after si-NC introduction, si-USP7 transfection, or si-USP7 transfection before MG132 exposure (100 nM, 24 h). F ) TPC-1 cells were co-transfected with Flag-FGFR1 plasmid and si-USP7 or sh-NC control, followed by immunoprecipitation (IP) experiment with anti-Flag antibody; the immunoprecipitates were subjected to immunoblot assay (IB) using anti-Ub antibody. G ) Representative immunofluorescence staining depicting the expression and localization of USP7 and FGFR1 in si-USP7-transfected or si-NC-introduced TPC-1 cells. * p <0.05.

Article Snippet: The primary antibodies, including rabbit anti-FGFR1 polyclonal (#GB115541, 1:100; Servicebio) and mouse anti-USP7 monoclonal (#66514-1-Ig, 1:300; Proteintech), were used prior to the application of Alexa 488-labeled anti-mouse (#GB25301, 1:500; Servicebio) and Cy3-labeled anti-rabbit (#GB21303, 1:500; Servicebio) IgG secondary antibodies following the vendors’ guidelines.

Techniques: Western Blot, Transfection, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Immunoprecipitation, Immunofluorescence, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: USP7 correlates with PTC cell malignant behaviors by stabilizing FGFR1. A ) Immunoblots of lysates of TPC-1 and SW579 PTC cells that were introduced with si-USP7, si-USP7+FGFR1 plasmid or scrambled siRNA sequence (si-NC) for evaluation of FGFR1 protein. B ) Viability analysis of TPC-1 and SW579 PTC cells transfected as in A by CCK-8 assay. C,D ) Proliferative ability evaluation of TPC-1 and SW579 PTC cells transfected as in A by EdU assay. E ) Apoptosis analysis of TPC-1 and SW579 PTC cells treated as in A by flow cytometry based Annexin V-FITC and PI staining. F,G ) Assessment of invasiveness of TPC-1 and SW579 PTC cells introduced as in A by transwell assay. H) Determination of migratory ability of TPC-1 and SW579 PTC cells introduced as in A by would healing assay. * p <0.05.

Article Snippet: The primary antibodies, including rabbit anti-FGFR1 polyclonal (#GB115541, 1:100; Servicebio) and mouse anti-USP7 monoclonal (#66514-1-Ig, 1:300; Proteintech), were used prior to the application of Alexa 488-labeled anti-mouse (#GB25301, 1:500; Servicebio) and Cy3-labeled anti-rabbit (#GB21303, 1:500; Servicebio) IgG secondary antibodies following the vendors’ guidelines.

Techniques: Western Blot, Plasmid Preparation, Sequencing, Transfection, CCK-8 Assay, EdU Assay, Flow Cytometry, Staining, Transwell Assay

Journal: European Journal of Histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: TF YY1 can promote FGFR1 transcription. A ) Schematic of the motif of YY1, the predicted pairing sequence for YY1 in the FGFR1 promoter, the mutation in the pairing sequence. B ) ChIP experiments of chromatin of TPC-1 and SW579 cells using antibodies against YY1 and IgG, followed by DNA purification and enrichment analysis of the FGFR1 promoter. C ) Immunoblots of lysates of si-YY1- or si-NC-transfected TPC-1 and SW579 cells to assess YY1 protein expression. D,E ) The reporter construct WT-FGFR1 or MUT-FGFR1 was introduced into TPC-1 and SW579 cells along with si-YY1 or si-NC control, followed by analysis of luciferase activity. F) Representative immunoblotting showing FGFR1 protein level in si-YY1- or si-NC-introduced TPC-1 and SW579 cells. * p <0.05.

Article Snippet: The primary antibodies, including rabbit anti-FGFR1 polyclonal (#GB115541, 1:100; Servicebio) and mouse anti-USP7 monoclonal (#66514-1-Ig, 1:300; Proteintech), were used prior to the application of Alexa 488-labeled anti-mouse (#GB25301, 1:500; Servicebio) and Cy3-labeled anti-rabbit (#GB21303, 1:500; Servicebio) IgG secondary antibodies following the vendors’ guidelines.

Techniques: Sequencing, Mutagenesis, DNA Purification, Western Blot, Transfection, Expressing, Construct, Control, Luciferase, Activity Assay

Journal: European Journal of Histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: Reduction of USP7 prevents xenograft growth by downregulating FGFR1. A,B ) Xenograft growth curve ( A ) and weight ( B ) in BALB/c nude mice after injection of sh-NC lentivirus-infected SW579 cells, sh-USP7 lentivirus-transduced SW579 cells with or without intratumor injection of FGFR1 expression plasmid. C ) Immunoblots of sh-NC SW579 xenografts, sh-USP7 SW579 xenografts and shUSP7+FGFR1 xenografts to evaluate FGFR1 protein expression. D ) Representative immunohistochemical staining of xenografts shown in C for evaluation of FGFR1 expression and the Ki67-positive cells. * p <0.05.

Article Snippet: The primary antibodies, including rabbit anti-FGFR1 polyclonal (#GB115541, 1:100; Servicebio) and mouse anti-USP7 monoclonal (#66514-1-Ig, 1:300; Proteintech), were used prior to the application of Alexa 488-labeled anti-mouse (#GB25301, 1:500; Servicebio) and Cy3-labeled anti-rabbit (#GB21303, 1:500; Servicebio) IgG secondary antibodies following the vendors’ guidelines.

Techniques: Injection, Infection, Expressing, Plasmid Preparation, Western Blot, Immunohistochemical staining, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma

doi: 10.4081/ejh.2024.4048

Figure Lengend Snippet: Schematic of the USP7/FGFR1 and YY1/FGFR1 cascades in PTC progression. In PTC cells, the TF YY1 promotes the transcription of FGFR1 and the deubiquitinating enzyme USP7 stabilizes FGFR1, thereby contributing PTC progression

Article Snippet: The primary antibodies, including rabbit anti-FGFR1 polyclonal (#GB115541, 1:100; Servicebio) and mouse anti-USP7 monoclonal (#66514-1-Ig, 1:300; Proteintech), were used prior to the application of Alexa 488-labeled anti-mouse (#GB25301, 1:500; Servicebio) and Cy3-labeled anti-rabbit (#GB21303, 1:500; Servicebio) IgG secondary antibodies following the vendors’ guidelines.

Techniques:

Journal: Cell Reports Medicine

Article Title: Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation

doi: 10.1016/j.xcrm.2020.100153

Figure Lengend Snippet: Generation and Characterization of hiPSCs Carrying the SERPINE1-FOSB Translocation (A) Schematic overview of the targeting strategy for generation of a SERPINE1-FOSB gene fusion. Filled boxes are exons, lines introns. FOSB start codons are labeled in the figure; black text represents the original start codon, while the new start codon after the fusion is shown in red. Two double-stranded breaks were introduced in the genome guided by 2 gRNAs in SERPINE1 intron 1 and FOSB intron 1. A repair template used for homologous recombination (HR template) with neomycin resistance cassette flanked by Flp-recombinase sequences (FRTs), as well as targeted genomic locus before (After HR) and after FLP-mediated neomycin removal (after FLPO). The bottom panel shows Sanger sequencing of PCR products from the clone with translocation validating HDR recombination of SERPINE1 and FOSB , with the remaining FRT sequence left from the repair template (D3 clone). (B) Representative results of PCR screen on single-cell-derived hiPSC clones using primers (F2, R2 and SF, SR; F2, SR and FF and FR) shown in the panel above the PCR screen results. Two targeted clones (D3 and G6) were identified of 73 screened clones. PCR shows that clone G6 has a large insert in the SERPINE1 wild-type allele. (C) COBRA-FISH on colony metaphase cells of WT, D3, and G6 hiPSC clones shows a balanced translocation t(7;19)(q22;q13); furthermore, no additional chromosomal abnormalities were evident in any of the screened cells. (D) Whole-genome sequencing was performed, and the results are summarized in a Circos plot. The first layer shows all genes that are potential off-target sites for the gRNA for FOSB (red) and SERPINE1 (blue). No mutations were found in the off-target sites and the surrounding 100 bases. The second and third layers show copy number analysis (CNA) for clones D3 and G6, respectively, compared to the isogenic control. No copy number variations (CNVs) are detected. The green connection line shows the detected SERPINE1-FOSB fusion, as detected in both clones D3 and G6. Chromosomes 7 and 19, involved in the translocation, are highlighted in red. (E) Teratoma formation in mice. The top panel shows teratomas formed from the hiPSC WT , the bottom panel from the hiPSC SERPINE1-FOSB (D3) ; 2 sections of each are shown. Cellular derivatives of the 3 germ lineages are indicated: mesoderm (MES), ectoderm (ECT), and endoderm (END). Scale bar indicates 200 μm.

Article Snippet: Western blotting was performed as previously described using FOSB monoclonal rabbit antibody (#2251; Cell Signaling) and USP7 monoclonal rabbit antibody (A300-033A; Bethyl).

Techniques: Translocation Assay, Labeling, Homologous Recombination, Sequencing, Derivative Assay, Clone Assay, Combined Bisulfite Restriction Analysis Assay, Control

Journal: Cell Reports Medicine

Article Title: Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation

doi: 10.1016/j.xcrm.2020.100153

Figure Lengend Snippet: hiPSC-ECs Carrying the SERPINE1-FOSB Translocation Show Increased FOSB Expression (A) Schematic overview of the differentiation protocol and purification of ECs from hiPSCs. (B) Bright-field images showing typical EC morphology of hiPSC-ECs. Scale bar represents 500 μm. (C) Fluorescence-activated cell sorting (FACS) analysis of EC marker expression on isolated ECs at passage 3 (P3) from hiPSC-ECs WT (black-filled histogram) and hiPSC-ECs SERPINE1-FOSB (D3) (red-filled histogram), and relevant isotype control (gray-filled histogram). (D) Quantification of normalized relative surface expression levels (MFI) of VEC, VEGFR2, VEGFR3, CD31, CD34, and CD105. n = 3 (biological replicates, 3 independent batches of hiPSC-ECs). Error bars are SDs, ∗p < 0.005. (E) Real-time qPCR analysis of FOSB expression in hiPSCs WT , hiPSCs SERPINE1-FOSB (D3) , hiPSC-ECs WT , and hiPSC-ECs SERPINE1-FOSB (D3) normalized to the housekeeping gene HPRT1 (×1,000). n = 3 (biological replicates, 3 independent batches of hiPSC-ECs). Error bars represent means ± SDs. (F) Western blot of FOSB expression in hiPSC-ECs WT and hiPSC-ECs SERPINE1-FOSB (D3) . Short and long exposure of the gel is shown. USP7 was used as a housekeeping control.

Article Snippet: Western blotting was performed as previously described using FOSB monoclonal rabbit antibody (#2251; Cell Signaling) and USP7 monoclonal rabbit antibody (A300-033A; Bethyl).

Techniques: Translocation Assay, Expressing, Purification, Fluorescence, FACS, Marker, Isolation, Control, Western Blot

Journal: Cell Reports Medicine

Article Title: Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation

doi: 10.1016/j.xcrm.2020.100153

Figure Lengend Snippet: Transcriptome Analysis of hiPSC-ECs Carrying the SERPINE1-FOSB Translocation (A) Hierarchical clustering analysis (HCA) of differentially expressed genes (DEGs) between hiPSC-ECs WT (WT) and hiPSC-ECs SERPINE1-FOSB (D3) (MUT) samples (3 independent differentiations and isolation for each clone). A total of 630 and 592 significantly upregulated and downregulated genes in MUT were identified compared to WT ECs (p FDR ≤ 0.05). (B) Representative KEGG pathways enriched in DEGs upregulated in MUT ECs (−log10(adjusted p value)) and number of enriched genes within total genes of each pathway are shown. (C) Representative Gene Ontology (GO) enriched in DEGs upregulated in WT or DEGs upregulated in MUT ECs. Size and color indicate gene number and adjusted p value of each GO. (D) Heatmaps of genes from GOs enriched in hiPSC-ECs SERPINE1-FOSB(D3) upregulated DEGs. (E) Gene interaction network of genes from GOs shown in (C and D and Figure S4 C) constructed using Ingenuity Pathway Analysis (IPA). SERPINE1 and FOSB were added manually. Interactions among FOSB and TGF-β signaling pathway and 2 networks were generated using IPA. Color indicates the log2(fold change) of gene expression in MUT compared to WT.

Article Snippet: Western blotting was performed as previously described using FOSB monoclonal rabbit antibody (#2251; Cell Signaling) and USP7 monoclonal rabbit antibody (A300-033A; Bethyl).

Techniques: Translocation Assay, Isolation, Construct, Generated, Gene Expression

Journal: Cell Reports Medicine

Article Title: Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation

doi: 10.1016/j.xcrm.2020.100153

Figure Lengend Snippet: Functional Assessment of hiPSC-ECs Carrying the SERPINE1-FOSB Translocation (A) Analysis of hiPSC-ECs WT and hiPSC-ECs SERPINE1-FOSB (D3) proliferation rates when cultured in basal endothelial cell growth medium supplemented with 1% PPS (1%), 1% PPS supplemented with 50 ng/mL VEGF (1% VE), or complete EC growth medium (full) for 24 h. Proliferation was determined by using a Presto Blue assay. n = 3 (biological replicates, 3 independent batches of hiPSC-ECs). Error bars are shown as SDs; ∗p < 0.0001 and ∗∗p < 0.0005. (B) Representative images of Matrigel tube formation assay using hiPSC-ECs WT and hiPSC-ECs SERPINE1-FOSB (D3) at the 48-h time point. Scale bar represents 500 μm. The right panel shows the quantification of the number of junctions and meshes. Error bars are SDs; ∗p < 0.005 and ∗∗p < 0.001. (C) Representative absolute resistance of the EC monolayer in complete EC growth medium. N = 6 (2 independent experiments with 3 batches of hiPSC-ECs). Errors bars are shown as means ± SDs. (D) Normalized resistance (4 kHz) of the EC monolayer in complete EC growth medium. N = 6 (2 independent experiments with 3 batches of hiPSC-ECs). Error bars are shown as means ± SDs; ∗∗∗p < 0.001. (E) Representative immunofluorescent images of CD31 and ZO1 to analyze the cell tight junctions. Merged images show CD31 in red, ZO1 in green, and DAPI in blue. The right panels show further enlarged areas selected from the shown images (dashed squares). Scale bar represents 50 μm.

Article Snippet: Western blotting was performed as previously described using FOSB monoclonal rabbit antibody (#2251; Cell Signaling) and USP7 monoclonal rabbit antibody (A300-033A; Bethyl).

Techniques: Functional Assay, Translocation Assay, Cell Culture, Tube Formation Assay

Journal: Cell Reports Medicine

Article Title: Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation

doi: 10.1016/j.xcrm.2020.100153

Figure Lengend Snippet: In Vivo Vasculogenesis Assay for hiPSC-ECs WT and hiPSC-ECs SERPINE1-FOSB (D3) (A and C) H&E and human CD31 staining of FFPE tissue from the Matrigel Plug harvested after 4 (A) and 16 (C) weeks. Both hiPSC WT and hiPSC SERPINE-FOSB (D3) show vessel formation. Scale bar indicates 100 μm. (B and D) Vessel density was estimated by quantification of the human CD31 + area at 4 and 16 weeks. The 4- and 16-week time points showed no significant difference in human CD31 + area. Error bars are shown as SDs; ns, not significant. (E and F) Double immunofluorescent staining with FOSB and human CD31 antibodies counterstained with DAPI on cryosections from Matrigel Plug Assay containing hiPSC-ECs WT and hiPSC-ECs SERPINE-FOSB (D3) . FOSB is shown in red, CD31 in green, and DAPI in blue. The left panel shows the hiPSC-ECs WT experiment, and the right panel shows the hiPSC-ECs SERPINE-FOSB (D3) experiment. Cyan-colored objects represent erythrocytes fluorescing in multiple detection channels. Scale bar indicates 100 μm. (G) FOSB immunohistochemistry (IHC) on FFPE tissue from Matrigel Plug with hiPSC SERPINE-FOSB (D30) ECs. Scale bar, 50 μm. Surrounding mouse muscle (indicated by M) and the Matrigel Plug (indicated by P). (F) and (G) show the invasion of FOSB + hiPSC-ECs SERPINE1-FOSB (D3) into the striated muscle at 16 weeks post-transplantation. (H and I) PTAH-stained sections from the in vivo vasculogenesis assay. hiPSC-ECs WT (left panel) and hiPSC-ECs SERPINE-FOSB (D3) (right panel) are shown. Both images show the surrounding mouse muscle (indicated by M) and the Matrigel Plug (indicated by P). Vessels with and without thrombi are indicated by red and green arrows, respectively (H), and red and green stars, respectively (I). The black arrows indicate areas with infiltration in the mouse muscle. The scale bar indicates 50 μm. (J) Quantification of vessels containing PTAH + thrombi in hiPSC-ECs WT and hiPSC-ECs SERPINE-FOSB (D3) , in an area of 5.7 mm 2 . Error bars are shown as SDs; n = 3 and p = 0.1.

Article Snippet: Western blotting was performed as previously described using FOSB monoclonal rabbit antibody (#2251; Cell Signaling) and USP7 monoclonal rabbit antibody (A300-033A; Bethyl).

Techniques: In Vivo, Staining, Matrigel Assay, Immunohistochemistry, Transplantation Assay

Journal: Cell Reports Medicine

Article Title: Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation

doi: 10.1016/j.xcrm.2020.100153

Figure Lengend Snippet:

Article Snippet: Western blotting was performed as previously described using FOSB monoclonal rabbit antibody (#2251; Cell Signaling) and USP7 monoclonal rabbit antibody (A300-033A; Bethyl).

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, DNA Purification, Gene Expression, Sequencing, Plasmid Preparation, Software