Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Immunoblot analysis of Jurkat NF-κB-GFP reporter cells lentivirally transduced with empty vector or with viruses driving expression of wild type TRAF3IP2-V5 or the FYN-TRAF3IP2-V5 fusion. Expression levels were verified in 3 independent experiments. b, NF-κB-GFP reporter activity in Jurkat cells as in a after stimulation with anti-CD3 (a-CD3) antibody. Results are reported as mean of fold change over empty vector IgG control from n=3 independent experiments ± s.d. Individual values are shown as white circles. P values were calculated using two-way ANOVA and Tukey’s or Sidak’s multiple comparison test. c, NF-κB-GFP reporter activity in Jurkat cells as in a after treatment with increasing doses of PMA. Bar graphs indicate the mean of technical replicate values obtained in parallel. Individual values are shown as white circles. The experiment was performed once.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Western Blot, Transduction, Plasmid Preparation, Expressing, Activity Assay

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: FYN-TRAF3IP2 signaling requires membrane localization. a, Schematic representation of the FYN-TRAF3IP2 fusion protein indicating the wild type and mutant membrane localization motif sequences. b, Immunoblot analysis (top) and quantification (bottom) of cytosolic and membrane-associated FLAG-tagged proteins after subcellular fractionation in Jurkat cells expressing wild type TRAF3IP2–3xFLAG, FYN-TRAF3IP2–3xFLAG, or membrane localization mutant ASVQSKDK FYN-TRAF3IP2–3xFLAG. Expression levels and fractionation controls were verified for each independent experiment. IB, immunoblot. c, Immunofluorescence analysis of cellular localization of GFP-tagged proteins in Karpas-299 cells expressing FYN-TRAF3IP2-GFP, and membrane localization mutant ASVQSKDK FYN-TRAF3IP2-GFP. GFP-tagged proteins are shown in green, the Na/K ATPase membrane marker in purple, and DAPI-stained nuclei in blue. The experiment was performed twice with similar results. Scale bar represents 10 μm. d, NF-κB dual luciferase reporter activity (right) and immunoblot analysis (left) of 293T cells transfected with empty vector or lentivirus driving expression of wild type TRAF3IP2-V5, FYN-TRAF3IP2-V5, or mutant ASVQSKDK FYN-TRAF3IP2-V5. Bar graphs indicate the mean of fold change over empty vector from n=2 independent experiments. Individual values are shown as white circles.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Mutagenesis, Western Blot, Fractionation, Expressing, Immunofluorescence, Marker, Staining, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a. We transduced hematopoietic progenitors from CD4 Cre-ERT2 Tet2fl/fl mice with bicistronic retroviruses driving the expression of GFP, FYN-TRAF3IP2-V5 and GFP or wild type TRAF3IP2-V5 and GFP. We intravenously transplanted the infected cells into lethally irradiated C57BL/6 recipient mice and treated them 8 weeks post-transplant with vehicle only or with tamoxifen to preserve or delete Tet2 in CD4+ T cells, respectively. These cohorts of mice were then immunized with sheep red blood cells every 4–5 weeks to induce peripheral T cell activation. b, Representative histological micrographs of hematoxylin-eosin stained liver, lung, and kidney of Tet2fl/fl or Tet2−/− FYN-TRAF3IP2-induced lymphoma-bearing animals and the GFP only control. Tissues from 3 mice per group showed similar results. Scale bar = 200 μm. c, Representative FACS plots of mononuclear cells collected from liver, lung, and kidney of FYN-TRAF3IP2-induced lymphoma-bearing animals, showing GFP+ CD4+ cell infiltration. d, Quantitative analysis of lymphoma liver infiltration in FYN-TRAF3IP2 Tet2fl/fl and FYN-TRAF3IP2 Tet2−/− tumors. Results are reported as mean of values (bar) ± standard deviation (error bar) with individual values (white circles), n=5 (5 tumors from 5 different mice per condition). The P value was calculated using two-tailed‎ Student’s‎ t-test. e, Representative flow cytometry analyses of PD1, ICOS, BCL6 and CXCR5 Tfh cell marker expression in CD4+ GFP+ spleen tumor cells compared to CD4+ GFP− non-tumor cells from the same spleen. f, Flow cytometry analysis of PD1 and CXCR5 Tfh cell marker expression in CD4+ GFP− non-tumor cells and CD4+ GFP+ tumor cells from a representative FYN-TRAF3IP2-induced lymphoma-bearing spleen. g, Heatmap representation of Tfh-associated marker expression in CD4+ naïve wild type T cells, FYN-TRAF3IP2-induced CD4+ GFP+ lymphoma cells and RHOA G17V Tet2−/− AITL-like mouse tumor cells53. h, GSEA enrichment plots of differentially expressed genes associated with FYN-TRAF3IP2-induced mouse lymphoma cells compared to wild type naïve CD4+ T cells. AITL geneset: top differentially upregulated genes in AITL compared with PTCL, NOS (fold change 1.5, p <0.002)38. Tfh geneset: top 100 genes associated with Tfh cells37.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Expressing, Infection, Irradiation, Activation Assay, Staining, Standard Deviation, Flow Cytometry, Marker

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Kaplan-Meier survival curves of mice transplanted with bone marrow progenitor cells from CD4 Cre-ERT2 Tet2fl/fl mice retrovirally transduced to express GFP only, wild type TRAF3IP2 and GFP, or fusion FYN-TRAF3IP2 and GFP treated with vehicle (blue arrow) 8 weeks post-transplant. 10 mice were transplanted per experimental group (n=number of mice). Black arrows indicate the red blood cell immunizations. b, Splenic weight of animals at the endpoint after transplantation as in a. Red circles, GFP+ CD4+ lymphoma; black circles, no lymphoma; gray circle, GFP+ CD8+ lymphoma; horizontal line, mean value. P values were calculated using two-tailed‎ Student’s‎ t-test. c, Kaplan-Meier survival curves of mice transplanted with bone marrow progenitor cells from CD4 Cre-ERT2 Tet2fl/fl mice retrovirally transduced to express GFP only, wild type TRAF3IP2 and GFP, or fusion FYN-TRAF3IP2 and GFP treated with tamoxifen (red arrow) 8 weeks post-transplant. 10 mice were transplanted per experimental group (n=number of mice). Black arrows indicate the red blood cell immunizations. d, Splenic weight of animals at the endpoint after transplantation as in c. Red circles, GFP+ CD4+ lymphoma; black circles, no lymphoma; horizontal line, mean value. P values were calculated using two-tailed‎ Student’s‎ t-test. e, Spleens from FYN-TRAF3IP2-induced lymphoma-bearing mice (bottom) and nine representative mice that were lymphoma-free at the end of follow up (top) and representative lymph nodes from FYN-TRAF3IP2-induced lymphoma-bearing mice. f, Representative FACS plots showing GFP+ CD4+ FYN-TRAF3IP2 lymphoma cells infiltrating lymph nodes and bone marrow. g, Histological micrographs of hematoxylin-eosin stained spleen sections of immunized control mice and FYN-TRAF3IP2-induced CD4+ PTCL, NOS. Tissues from 3 mice per group showed similar results. Scale bar = 400 μm.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Transplantation Assay, Staining

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Immunofluorescence analysis of p65 NF-κB intracellular localization in naïve CD4+ T cells, in normal GFP+ CD4+ cells from mice transplanted with Tet2−/− progenitors infected with GFP expressing retroviruses, in GFP+ Tet2f/f FYN-TRAF3IP2 CD4+ lymphoma cells and in GFP+ Tet2−/− FYN-TRAF3IP2 CD4+ lymphoma. p65 proteins shown in red and DAPI-stained nuclei in blue. 2 representative images per condition are shown. Scale bar = 5 μm. b, Quantitation (20 cells from 1 mouse per group) of p65 NF-κB intracellular nuclear/cytoplasmic localization in CD4+ cells as in a. N/C ratio, nuclear/cytoplasmic ratio. The boxplot lines represent median, boxes represent 25–75% intervals, whiskers represent 10–90% intervals and circles represent data points outside the 10–90% interval. P values were calculated using one-way ANOVA and Tukey’s multiple comparison test. c, Flow cytometry analysis of phospho-p65 NF-κB transcription factor levels in FYN-TRAF3IP2-induced CD4+ GFP+ lymphoma cells compared to wild type CD4+ T cell controls. Horizontal lines in the graph indicate the mean, and circles represent values for six independent tumors (red; two Tet2fl/fl FYN-TRAF3IP2 and four Tet2−/− FYN-TRAF3IP2 lymphomas) and three normal CD4+ T cell control samples (black). The P value was calculated using two-tailed‎ Student’s‎ t-test. AF647, Alexa Fluor 647. d, Western blot analysis and quantitation of canonical p105 and alternative p100 NF-κB factor precursor cleavage into active p52 and p50 forms in Tet2−/− FYN-TRAF3IP2 lymphoma cells. Results are reported as mean of n=2 independent experiments with individual values shown as white circles. Expression levels were verified for each independent experiment. e, Bar graph representation of enrichment scores for NF-κB gene signatures obtained by Gene Set Enrichment Analysis (GSEA) of differentially expressed genes associated with FYN-TRAF3IP2-induced mouse CD4+ PTCL, NOS. BCL, B-cell lymphoma. f, GSEA plot corresponding to analysis of the NF-κB target geneset of the BU Gilmore lab database as in e. g, Heat map representation of the GSEA leading edge genes from the analysis of the NF-κB target geneset of the BU Gilmore lab database as in e, f. Scale bar shows color-coded differential expression, with red indicating higher levels of expression and blue indicating lower levels of expression.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Immunofluorescence, Infection, Expressing, Staining, Quantitation Assay, Flow Cytometry, Western Blot

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, In vitro cell viability relative to vehicle only (DMSO) controls of FYN-TRAF3IP2-induced mouse lymphoma. Data are shown as mean of technical replicates. The experiment was performed with 3 independent tumors from different mice. b, Flow cytometry analysis of cell cycle in FYN-TRAF3IP2-induced mouse lymphoma cells treated in vitro with DMSO and IC50 concentration of IKK beta inhibitors BMS-345541 (5 μM) and IKK-16 (2 μM). Results are reported as mean of technical replicate values from 1 tumor per condition (bar) with individual values shown as white circles. c, Representative FACS plots and quantitation of apoptosis as in b. d, Luciferase in vivo bioimaging analysis of response of FYN-TRAF3IP2-induced mouse lymphomas treated with vehicle or BMS-345541 (n=10 mice in the vehicle group and n=9 mice in the treatment group). Graph indicates fold changes in bioluminescence relative to the basal signal before treatment. e, Kaplan-Meier survival curve of the mice treated with BMS-345541 or vehicle as in d. The P value was calculated using the Log-rank (Mantel-Cox) test. f, Spleen weight of FYN-TRAF3IP2-induced lymphoma-bearing mice treated with vehicle only or BMS-345541 at the endpoint as in d, e. g, Splenic tumor burden as in f. h, Immunofluorescence analysis and quantitation (20 cells from 1 mouse per condition) of p65 NF-κB nuclear/cytoplasmic localization in FYN-TRAF3IP2-induced mouse lymphoma cells after treatment with vehicle or BMS-345541 in vivo. p65 protein is shown in red and DAPI-stained nuclei in blue. Scale bar = 5 μm; N/C ratio, nuclear/cytoplasmic ratio. The boxplot lines indicate the median, boxes represent 25–75% intervals, whiskers represent 10–90% intervals and circles represent data points outside the 10–90% intervals. i, Immunohistochemistry analysis of cell proliferation (Ki67) and apoptosis (cleaved caspase-3) in spleen sections of FYN-TRAF3IP2-induced lymphoma-bearing mice after treatment with vehicle only or BMS-345541. Tissues from 3 mice per group showed similar results. Scale bar = 100 μm. Horizontal lines in graphs shown in d, f, and g indicate mean and circles correspond to individual values. P values in d, f, g and h were calculated using two-tailed‎ Student’s‎ t-test. P value in e was calculated using the log-rank test.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: In Vitro, Flow Cytometry, Concentration Assay, Quantitation Assay, Luciferase, In Vivo, Immunofluorescence, Staining, Immunohistochemistry

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, RNA expression level of IL17RC, essential for a functional IL17 receptor68, in T cells and monocytes represented from DICE (Database of Immune Cell Expression, Expression quantitative trait loci and Epigenomics). b, NF-κB-GFP reporter activity in transduced Jurkat cells after stimulation with 200 ng/mL IL17A. Results are reported as mean of technical replicate values (bar) from 1 independent experiment with individual values (white circles), n=1. c, Immunoblot analysis of JPM50.6 CARD11 knockout and JPM50.6 cells reconstituted by CARD11-HA expression. Expression levels were verified for each independent experiment. d, NF-κB-GFP reporter activity after stimulation with 25 nM of PMA or 20 ng/mL TNFα as in c. e, Western blot analysis of FYN-TRAF3IP2-V5 and BCL10 expression in Jurkat cells and Jurkat BCL10 knockout cells infected with empty vector or FYN-TRAF3IP2-V5 expressing lentiviruses; and RT-PCR analysis of FYN-TRAF3IP2-V5 mRNA in the same Jurkat BCL10 knockout cells infected with empty vector or FYN-TRAF3IP2-V5 expressing lentiviruses. * and ** indicate non-specific bands detected by the V5 antibody, while the arrowhead indicates the location of the specific FYN-TRAF3IP2-V5 band detected with the same antibody. Expression levels were verified for each independent experiment.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Expressing, Functional Assay, RNA Expression, Activity Assay, Western Blot, Knock-Out, Infection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Kaplan-Meier survival curve of 9 mice transplanted with cell suspension containing FYN-TRAF3IP2 GFP+ lymphoma infiltrate. b, Spleens of secondary recipients transplanted with FYN-TRAF3IP2-induced lymphoma infiltrate. c, Representative histological micrographs of H&E stained spleen, liver, and lung of lymphoma transplanted mice at the endpoint showing lymphoma infiltration. Tissues from 3 mice showed similar results. Scale bar = 400 μm. d, Representative FACS plot showing GFP+ CD4+ lymphoma infiltrates in spleens from diseased secondary recipients. e, Representative flow cytometry analyses of PD1, ICOS and BCL6 Tfh cell marker expression in CD4+ GFP+ spleen tumor cells compared to CD4+ GFP− non-tumor cells from the same spleen. f, Tcrb gene clonal analysis of three secondary Tet2−/− FYN-TRAF3IP2 GFP+ lymphomas illustrated by side-by-side representation of Tcrb sequence reads from primary and secondary Tet2−/− FYN-TRAF3IP2 GFP+ lymphomas, indicating the retention and expansion of specific lymphoma clone with unique Tcrb rearrangements after transplantation. g, RNAseq data of six independent FYN-TRAF3IP2-induced mouse CD4+ GFP+ lymphomas and wild type isogenic mouse naïve CD4+ T cells represented as heat map of the unselected top 100 differentially expressed genes between FYN-TRAF3IP2-induced tumors and wild type CD4+ naïve T cells (mouse genes without known human orthologues excluded; scale bar shows color-coded differential expression, with red indicating higher levels of expression and blue indicating lower levels of expression). h, Volcano plot of all differentially expressed genes as in g (P value <0.001 and |log2(fold change)|> 1 deemed significant). Blue, significantly downregulated genes. Red, significantly upregulated genes. The number of significantly downregulated or upregulated genes is indicated. i, Principal component analysis plot of RNAseq data as in g. j-k, GSEA analyses of differentially expressed genes associated with FYN-TRAF3IP2-induced mouse lymphomas based on the RNAseq data and the Hallmark signatures69 from MSigDB. The top 6 upregulated signatures j and top 6 downregulated signatures k are represented as bar graphs of normalized enrichment scores and P values.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Staining, Flow Cytometry, Marker, Expressing, Sequencing, Transplantation Assay

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Representative flow cytometry plot and quantification of the analysis of apoptosis in normal CD4+ T cells and CD4 FYN-TRAF3IP2 lymphoma cells treated with vehicle only (DMSO) or the IKK16 inhibitor. b, Representative flow cytometry plot and quantification of analysis of apoptosis in normal CD4+ T cells and CD4 FYN-TRAF3IP2 lymphoma cells treated with vehicle only (DMSO) or the BMS345541 IKK inhibitor. c, Representative flow cytometry plot and quantification of normal lymphocytes in spleen and lymph nodes of mice treated with vehicle only or with the BMS-345541 IKK inhibitor in vivo. Results are reported as mean of replicate values from 3 mice (bar) ± standard deviation (error bar) with individual values (black circles), n=3. P values were calculated using two-tailed‎ Student’s‎ t-test.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Flow Cytometry, In Vivo, Standard Deviation

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Schematic representation of the FYN-TRAF3IP2 fusion transcripts identified in RNAseq. Each horizontal line represents a chimeric FYN-TRAF3IP2 RNAseq read. b, Representative dideoxynucleotide sequencing result of the FYN-TRAF3IP2 cDNA from a PTCL index sample. c, Frequency and distribution across PTCL groups of samples harboring the FYN-TRAF3IP2 fusion transcript identified by RT-PCR (total patients n=30; total samples n=31, AITL, n=9; PTCL, NOS, n=6 (includes a paired diagnostic-relapse pair from same patient); extranodal NKTCL, nasal type, n=4; anaplastic T cell lymphoma, ALCL, n=4; hepatosplenic T cell lymphoma, HSTCL, n=3; cutaneous T cell lymphoma, CTCL, n=3; adult T cell leukemia/lymphoma, ATL, n=1; subcutaneous panniculitis-like T-cell lymphoma, SPTCL, n=1). d, Schematic representation of the chromosomal rearrangement event resulting in expression of the FYN-TRAF3IP2 fusion RNA and breakpoint sequence identified by whole genome sequencing of an index (RNAseq and RT-PCR positive) sample. e, Schematic representation of the structures of the FYN kinase, TRAF3IP2 protein, and FYN-TRAF3IP2 fusion protein. SH3, Src homology 3 domain; SH2, Src homology 2 domain; TBM, TRAF-binding motif; HLH, Helix-loop-helix domain; SEFIR, SEF/IL-17R signaling domain.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Diagnostic Assay, Expressing, Binding Assay

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Reverse-transcription PCR (RT-PCR) amplification results of an independent panel of 31 PTCL RNA samples spanning the region of FYN-TRAF3IP2 fusion breakpoint. b, DNA sequencing chromatograms of the RT-PCR amplicons generated in a.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, DNA Sequencing, Generated

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Reverse-transcription PCR (RT-PCR) amplification results of a panel of 92 B cell malignancies RNA samples spanning the region of FYN-TRAF3IP2 fusion breakpoint. + indicates a PTCL positive control sample, - indicates a PTCL negative control sample. No PCR products corresponding to the size of the specific FYN-TRAF3IP2 amplicon were detected. b, DNA sequencing chromatogram of the RT-PCR amplicons generated in a for the positive control PTCL sample and in sample 66 which showed a weak band slightly smaller than the specific FYN-TRAF3IP2 product. Lack of priming in this sample ruled out the presence of a specific FYN-TRAF3IP2 RT-PCR product in sample 66.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Negative Control, DNA Sequencing, Generated

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a, Immunoblot analysis of CARD11 knockout Jurkat cells and CARD11-HA reconstituted CARD11 knockout cells infected with empty vector or lentiviruses driving expression of TRAF3IP2-V5 or the FYN-TRAF3IP2-V5 fusion. Expression levels were verified in 2 independent experiments. b, NF-κB-GFP reporter activity after stimulation with 25 nM PMA in Jurkat cells as in a. Results are shown as mean of fold change over empty vector non treated CARD11 KO cells from n=2 independent experiments with individual values (white circles). c, NF-κB-GFP reporter activity in two independently FYN-TRAF3IP2-V5 infected Jurkat cell cultures (A3 and D5) after stimulation with PMA in presence and absence of Compound 3, a specific MALT1 inhibitor. Results are reported as mean of fold change over empty vector DMSO control from n=3 independent experiments (bar) ± s.d. (error bar) with individual values (white circles). P values were calculated using unpaired two-tailed‎ Student’s‎ t-test. d, Western blot analysis and quantitation of the RELA/p65 NF-κB subunit nuclear translocation in BCL10 knockout Jurkat cells infected with empty vector or a FYN-TRAF3IP2-V5 expressing vector after PMA stimulation. Results are reported as mean of fold change over empty vector DMSO control from n=2 independent experiments with individual values (white circles). Expression levels were verified for each independent experiment. e, Schematic representation of the FYN-TRAF3IP2 fusion protein indicating the wild type and mutant TRAF6-binding motif. f, Western blot of immunoprecipitation experiment analyzing the interaction between HA-TRAF6 and V5-tagged proteins in 293T cells expressing wild type TRAF3IP2-V5, FYN-TRAF3IP2-V5 or PVAVAA FYN-TRAF3IP2-V5 or in the empty vector control. The experiment was performed twice with similar results. g, Immunoblot analysis of Jurkat cells transduced with empty vector or lentivirus driving expression of wild type TRAF3IP2-V5, fusion FYN-TRAF3IP2-V5, or mutant PVAVAA FYN-TRAF3IP2-V5 (top) and corresponding NF-κB-GFP reporter activity after stimulation with 25nM PMA (bottom). Bars show the mean of fold change over empty vector from n=3 independent experiments (bar) ± standard deviation (error bar) with individual values (white circles). P values were calculated using one-way ANOVA and Tukey’s multiple comparison test. h, Schematic representation of the TCR/FYN-TRAF3IP2/ NF-κB signaling axis in PTCL. IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Western Blot, Knock-Out, Infection, Plasmid Preparation, Expressing, Activity Assay, Quantitation Assay, Translocation Assay, Mutagenesis, Binding Assay, Immunoprecipitation, Transduction, Standard Deviation

Journal: Nature cancer

Article Title: FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma

doi: 10.1038/s43018-020-00161-w

Figure Lengend Snippet: a. Representative histopathologic micrographs of hematoxylin-eosin stained and immunohistochemistry analysis of T cells (CD3, CD4), B cells (B220), follicular dendritic cells (CD21) and vasculature (CD31) of spleen and lymph node of individual FYN-TRAF3IP2 Tet2fl/fl or Tet2−/− FYN-TRAF3IP2-induced lymphoma-bearing animals (n=3 in each group). Similar fields on consecutive sections are shown in rows 1–4 and rows 7–8. Scale bar and size are shown in the micrographs. b, Immunoblot analysis of FYN-TRAF3IP2-V5 protein expression in control splenocytes and in the lymphoma-infiltrated spleen of a FYN-TRAF3IP2 diseased mouse. c, Representative FACS plot showing GFP+ CD4+ lymphoma cells in lymphoma-infiltrated spleens from FYN-TRAF3IP2 diseased mice. d, Tcr Vβ clonality in FYN-TRAF3IP2 lymphoma cells (GFP+ CD4+) and normal CD4 T cells (GFP− CD4+) from a diseased mouse. e, Tcrb clonal distribution in FYN-TRAF3IP2-induced CD4+ primary tumors and in a representative allografted secondary lymphoma. Sectors represent the fraction of reads corresponding to individual Tcrb sequences.

Article Snippet: Plasmid and vectors We obtained pLX304-blast-V5 construct encoding TRAF3IP2 and CARD11 from the Broad Lentiviral Expression Library, pcDNA3 Flag- TRAF6 (#66929) from Addgene, and FUW-mCherry-Puro-Luc from 61 .

Techniques: Staining, Immunohistochemistry, Western Blot, Expressing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Heatmap of ≥2-fold downregulated genes in MOLM13 cells upon HOTTIP −/−; as determined by RNA-seq. (B) GSEA of downregulated genes after HOTTIP −/−; . (C) Overlap of TADs identified by Hi-C in WT and HOTTIP −/−; MOLM13 cells. The domain score of an altered TAD was normalized (quantile-normalization) by subtracting the mean of all TAD Hi-C signals. ANOVA was used to identify significantly altered TADs (Bonferroni-corrected p value < 0.05). (D) GO analysis of genes encompassed by the decreased TADs upon HOTTIP −/−; . (E) Hi-C interaction map at the CTNNB1 locus comparing WT and HOTTIP −/−; MOLM13 cells. (F) ATAC-seq analysis of CTNNB1 in WT and HOTTIP −/−; MOLM13 cells. (G and H) NG Capture-C analysis of CTNNB1 (G) or MYC (H) promoter interactions, CTCF ChIP-seq and HOTTIP ChIRP-seq in WT and HOTTIP −/−; MOLM13 cells. Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively.

Article Snippet: For generation of the catalytically dead RNaseH-fused dCas9, mutated RNaseH (D210N) (Addgene #111904) was subcloned into the pHR-dCas9 vector (Addgene #46911) as a Sbf1-BamHI restriction fragment. sgRNA targeting the CTNNB1 CBS-u2 was subcloned into the pLKO5.sgRNA.EFS.tRFP vector (Addgene #57824) and transduced along with the dCas9-RNaseH fusion vector into AML cells, and then GFP/RFP double positive cells were selected by FACS.

Techniques: RNA Sequencing, Hi-C, Capture-C, ChIP-sequencing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Heatmap of ≥2-fold upregulated genes in BM LSK cells from Hottip -Tg mice. (B) GSEA of upregulated genes in LSK cells upon Hottip activation. (C) Heatmap of CTCF binding, from ChIP-seq, and Hottip binding, from ChIRP-seq, at CTCF/ Hottip co-occupied sites in BM LK cells from WT and Hottip -Tg mice. (D) CTCF ChIP-seq and Hottip ChIRP-seq binding profiles at the Ctnnb1 locus in WT and Hottip -Tg LK cells. (E) Hi-C interaction maps at the Ctnnb1 locus in WT and Hottip -Tg BM LK cells. CTCF-bound TAD boundaries indicated by red arrows. (F) Overlap of WT and Hottip -Tg Hi-C signals from (E). (G) ATAC-seq analysis of Ctnnb1 in WT and Hottip -Tg BM LSKs. (H) tSNE visualization of BM LK cell subsets from Hottip -Tg (red) and WT (blue) mice by scRNA-seq. LT-HSC, ST-HSC, and MPP populations encompassed by blue circle. (I) Trajectory inference branches/clusters were generated based on the expression levels of lineage-associated genes in cell clusters (left) from WT and Hottip -Tg BM LK cells. Sub-population cell density analysis (right) correlated with the enriched cell number of each population. Higher cell densities shown in dark red. (J) The levels of Myc and Hoxa9 in each cell subset along HSC to MEP differentiation in WT and Hottip -Tg BM LK cells by scRNA-seq. The FDR-corrected p value ≤0.05 by binomial and hypergeometric test. (K) Relative cell numbers in each cell subset along HSC to MEP differentiation in WT and Hottip -Tg BM LK cells by scRNA-seq. The FDR-corrected p value ≤0.05 by binomial and hypergeometric test. (L) GO analysis of upregulated genes in LT- and ST-HSC populations upon Hottip activation by scRNA-seq.

Article Snippet: For generation of the catalytically dead RNaseH-fused dCas9, mutated RNaseH (D210N) (Addgene #111904) was subcloned into the pHR-dCas9 vector (Addgene #46911) as a Sbf1-BamHI restriction fragment. sgRNA targeting the CTNNB1 CBS-u2 was subcloned into the pLKO5.sgRNA.EFS.tRFP vector (Addgene #57824) and transduced along with the dCas9-RNaseH fusion vector into AML cells, and then GFP/RFP double positive cells were selected by FACS.

Techniques: Activation Assay, Binding Assay, ChIP-sequencing, Hi-C, Generated, Expressing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) CTCF ChIP-seq and HOTTIP ChIRP-seq-binding profiles at the upstream CBS s of CTNNB1 in WT and HOTTIP −/−; MOLM13 cells. (B) Schematic of probes used in EMSA. The G-4 sequence in HOTTIP , the C-rich HOTTIP -binding motif in the CBS-u2 sequence, and the CTCF-binding motif are shown in red, green, and yellow highlighting, respectively. (C) EMSA of a Cy5-labeled HOTTIP RNA probe (red) and a Cy3-labeled CTNNB1 CBS-u2 probe (green). Orange indicates R-loop formation. RNase A, RNase H, DNase I, and S9.6 antibody added as indicated. (D) Schematic of RIDP procedure (left). RIDP RT-qPCR of HOTTIP precipitated by probes targeting the template or non-template strand of the CBS-u2 site (in relation to the direction of CTNNB1 transcription) or targeting the negative control CBS at ACTB in WT, HOTTIP −/−; or RNase H treatment (right). Data presented as mean ± SD; ***p ≤ 0.001. (E) CTCF and cohesin-binding ChIP-seq, R-loop DRIP-seq, HOTTIP -binding ChIRP-seq and nascent RNA GRO-seq profiles at the CTNNB1 locus in WT and HOTTIP −/−; MOLM13 cells. (F) H3K4me3 and H3K27me3 ChIP-seq enrichment profiles at the CTNNB1 locus in WT and HOTTIP −/−; MOLM13 cells.

Article Snippet: For generation of the catalytically dead RNaseH-fused dCas9, mutated RNaseH (D210N) (Addgene #111904) was subcloned into the pHR-dCas9 vector (Addgene #46911) as a Sbf1-BamHI restriction fragment. sgRNA targeting the CTNNB1 CBS-u2 was subcloned into the pLKO5.sgRNA.EFS.tRFP vector (Addgene #57824) and transduced along with the dCas9-RNaseH fusion vector into AML cells, and then GFP/RFP double positive cells were selected by FACS.

Techniques: ChIP-sequencing, Binding Assay, Sequencing, Labeling, Quantitative RT-PCR, Negative Control

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Schematic of the CTNNB1 locus showing the locations of CBS s, sub-TADs, and TAD. (B) CTCF ChIP-qPCR analysis of the indicated sites in WT, CBS-u1 −/−; and CBS-u2 −/−; MOLM13 cells. (C) RT-qPCR analysis of the indicated transcripts in WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (D) Proliferation of WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (E) HOTTIP ChIRP-qPCR analysis of the indicated sites in WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (F) DRIP-qPCR analysis of the indicated sites in WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (G) NG Capture-C analysis of CTNNB1 promoter interactions in WT and CBS-u2 −/−; MOLM13 cells. Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively. Capture-C data were aligned with CTCF, H3K4me3, and H3K27me3 ChIP-seq profiles in the CTNNB1 locus in WT, HOTTIP −/−; , and CBS-u2 −/−; MOLM13 cells. (H) Kaplan-Meier survival curves of NSG mice transplanted with WT, CTNNB1 +/− , and CBS-u2 −/−; MOLM13 cells. n = 5. (I) hCD45 + cells chimerism in the BM and PB of NSG mice transplanted with WT, CTNNB1 +/− , and CBS-u2 −/−; MOLM13 cells. n = 3. (J) Kaplan-Meier survival curves of NSG mice transplanted with WT or CBS-u2 −/−; primary AML cells carrying MLL + (LPP4) or NPM1 C+ FLT3-ITD + (#974) mutations. n = 4. (K) hCD45 + cell chimerism in the BM of NSG mice transplanted with WT or CBS-u2 −/−; primary AML cells. Data in (B)–(F) and (H)–(K) are presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Article Snippet: For generation of the catalytically dead RNaseH-fused dCas9, mutated RNaseH (D210N) (Addgene #111904) was subcloned into the pHR-dCas9 vector (Addgene #46911) as a Sbf1-BamHI restriction fragment. sgRNA targeting the CTNNB1 CBS-u2 was subcloned into the pLKO5.sgRNA.EFS.tRFP vector (Addgene #57824) and transduced along with the dCas9-RNaseH fusion vector into AML cells, and then GFP/RFP double positive cells were selected by FACS.

Techniques: ChIP-qPCR, Quantitative RT-PCR, Capture-C, ChIP-sequencing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Schematic of the CTNNB1 locus. Shown is the CBS-u2 sequence with CTCF (yellow) and HOTTIP (green) motifs and the sgRNA target site (red) indicated. (B) RT-qPCR analysis of β-catenin and its target gene expression upon expression of dCas9-RNase H or dCas9-RNase H D210N with or without CBS-u2 -targeted sgRNA or exogenous β-catenin expression in MOLM13 cells. (C) Proliferation of WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (D) CTCF ChIP-qPCR analysis of the indicated sites in WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (E) HOTTIP ChIRP qPCR analysis of the indicated sites in WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (F) DRIP-qPCR analysis of the indicated sites in WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (G) NG Capture-C analysis of CTNNB1 promoter interactions upon expression of CBS-u2 RH-WT . Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively. Capture-C data were aligned with CTCF, H3K4me3, and H3K27me3 ChIP-seq profiles in the CTNNB1 locus in WT, HOTTIP −/−; , and CBS-u2 RH-WT MOLM13 cells. (H) Kaplan-Meier survival curves of NBSGW mice transplanted with WT, CBS-u2 RH-Mut , or CBS-u2 RH-WT MOLM13 cells. (I) Kaplan-Meier survival curves of NSG mice transplanted with CBS-u2 RH-WT or CBS-u2 RH-Mut OCI-AML3 cells. Mice were sacrificed when they were paralyzed due to the disease. Data in (B)–(F) and (H)–(I) are presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Article Snippet: For generation of the catalytically dead RNaseH-fused dCas9, mutated RNaseH (D210N) (Addgene #111904) was subcloned into the pHR-dCas9 vector (Addgene #46911) as a Sbf1-BamHI restriction fragment. sgRNA targeting the CTNNB1 CBS-u2 was subcloned into the pLKO5.sgRNA.EFS.tRFP vector (Addgene #57824) and transduced along with the dCas9-RNaseH fusion vector into AML cells, and then GFP/RFP double positive cells were selected by FACS.

Techniques: Sequencing, Quantitative RT-PCR, Targeted Gene Expression, Expressing, ChIP-qPCR, Capture-C, ChIP-sequencing

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: Ectopic expression of GRHL3 does not influence proliferation/survival rate and cell cycle profile of T24 cells ( A , B ) Forced stable expression of GRHL3 in T24 cells resulted in significantly higher transcript and protein expression levels compared to parental T24 cells and empty vector controls (pLX304). Three biological replicates were used for RT-qPCR experiments. *** indicates p -value < 0.001. ( C ) Calculated population doublings using Thiazolyl blue tetrazolium bromide (MTT) assays were comparable between GRHL3-overexpressing T24 cells, parental T24 cells and empty vector controls after 11 days in culture (T24 vs. T24 + pLX304 vs. T24+GRHL3: 23.92 vs. 25.19 vs. 25.40 population doublings (PD) at day 11; n.s). ( D ) Cell cycle analysis was performed by Propidium iodide (PI) staining and relative amounts of cells in the respective cell cycle phases are indicated. No difference could be observed in G1, S or G2/M phases between parental cells, empty vector controls and ectopic GRHL3-expressing cells (statistically n.s.).

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Cell Cycle Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: GRHL3 overexpression reduces migration in scratch wound assay. ( A ) Representative scratch wound experiment, displaying the original scratch wound gap in each picture by red lines during gap closure over 12 h. ( B ) Quantification of the “wound” closure shows that ectopic GRHL3-expressing T24 cells migrate significantly more slowly compared to empty vector controls (T24 + pLX304) at 6, 9 and 12 h ( p = 0.0013 at 9 h). Seven replicates were performed in this experiment. ** indicates p -value < 0.01.

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Over Expression, Migration, Scratch Wound Assay Assay, Expressing, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: Ectopic overexpression of GRHL3 impairs invasion capacity of T24 cells in Boyden chamber assay. ( A ) Representative images from Boyden chamber assays showing “invaded” T24 cells stained with crystal violet, indicating lower invasive potential in GRHL3-overexpressing cells. Scale bar, 200 µm. ( B ) Analysis of the cell-covered area using software demonstrated that GRHL3-overexpressing cells (T24 + GRHL3) invaded through membranes to a significantly lower extent than empty vector controls (T24 + pLX304; p = 0.0317). SD bars are shown for a total of 70 images analyzed. * indicates p -value < 0.05.

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Over Expression, Boyden Chamber Assay, Staining, Software, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: (( Aa )–( Ae )) Representative formalin-fixed, paraffin-embedded (FFPE) hematoxylin and eosin (H&E) tissue sections of porcine bladders after 10 days in organ culture. ( Aa ) Native, untreated porcine bladder with intact morphology maintaining urothelium (black arrow), stroma and muscle tissue. ( Ab ) The de-epithelialized porcine bladder with stromal and muscle tissue, without urothelial re-growth, used as negative control. (( Ac )–( Ae )) Cell lines were seeded onto de-epithelialized bladder tissue and grown in organ culture. While non-invasive RT4 cells formed a stratified multi-layer on top of the basal membrane ( Ac , black arrow), T24 cells with empty vector controls (T24 + pLX304) showed diffuse invasion into the stromal compartment ( Ad , black dotted arrow). In contrast, T24 cells overexpressing GRHL3 (T24 + GRHL3) did not invade but instead formed a superficial multi-layer on the pig stroma, similar to non-invasive RT4 cells ( Ae ). Four biological replicates were performed for each experiment. Scale bar, 200 µm. ( B ) Anti-human leukocyte antigen (Anti-HLA) staining confirms the invasive capacity of T24 + pLX304 cells into the pig bladder stroma and muscle tissue as well as the reduced invasion ability upon overexpression of T24 + GRHL3. (H&E, hematoxylin and eosin; NC, negative control).

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Formalin-fixed Paraffin-Embedded, Organ Culture, Negative Control, Membrane, Plasmid Preparation, Staining, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: Ectopic expression of GRHL3 drives T24 cells towards a more differentiated phenotype when cultured in organ culture conditions allowing cell–stroma interactions. ( A ) Native porcine urothelium expresses FOXA1 in organ culture. ( B ) De-epithelialized porcine bladder was used as s negative control. ( C ) RT4 cells seeded on the de-epithelialized porcine bladder form a multi-layered epithelial lining. Not all cells are FOXA1-positive (red arrow), indicating partial differentiation. ( D ) T24+pLX304 cells invade into the porcine bladder stroma and muscle and lack FOXA1 expression (black dotted arrows). ( E ) GRHL3-expressing T24 cells do not invade and express FOXA1 (red dotted arrow). ( F ) Western blot shows FOXA1 protein expression only in RT4 but not in T24 cells, irrespective of ectopic GRHL3 expression when grown in 2D culture conditions. ( G ) Similarly, FOXA1 expression in 2D cell culture is not upregulated in T24 + GRHL3 cells. *** indicates p -value < 0.001; ns = not significant.

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Expressing, Cell Culture, Organ Culture, Negative Control, Western Blot