Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: Sp1 is deacetylated by HDAC1/2/6 in TMZ-resistant GBM cells. (A) The wild-type (Wt) and TMZ-resistant (TMZ-R) GBM cells,12 as well as GBM spheroids formed in serum-free medium/suspension (S/S) culture and the control attached (Adh) cells13 were used for the immunoprecipitation (IP) assay with rabbit IgG, anti-Sp1 (Sp1, panel a), and anti–acetyl-lysine (ac-K, panels b and c) antibodies, and analyzed using immunoblotting (IB) as indicated. In panel c, the protein level of acetylated Sp1 was normalized to its total protein and quantified. (B) Gene expression profiles of HDACs in brain tumors were analyzed using the Oncomine database. HDAC1/3/6/9, shown by the arrows, were upregulated more in cancer tissues than in normal samples. Red indicates upregulation; blue indicates downregulation. The number in the cell represents the number of datasets that pass the filter criteria (threshold: P < 0.05). (C to F) Cells were harvested and analyzed using IB. The Wt (C and E) and TMZ-R (E) A172 cells were treated with the indicated concentrations of TMZ for 3 days. (D) The protein expression of HDACs in Wt and TMZ-R P11 GBM cells was normalized to the loading control and quantified. (F) The levels of HDAC6 and tubulin acetylation in attached GBM cells and tumorspheres. (G) Wt and TMZ-R P11 cells were used for IP assay with anti-Sp1 antibodies and rabbit IgG, and analyzed using IB as indicated. (H) TMZ-R U87MG cells were transfected with a nontargeting control siRNA or HDAC1/2/6-specific siRNAs as indicated. After knockdown, the cells were used for IP assay. (t-test: *P < 0.05, ***P < 0.001)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Suspension, Control, Immunoprecipitation, Western Blot, Gene Expression, Expressing, Transfection, Knockdown

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: HDAC1/2/6 inhibition significantly reduces the growth rates of TMZ-resistant GBM cells. (A) U87MG cells, as well as primary cultures of neurons and glial cells, were treated with 1 μM SAHA (SA), 1 μM azaindolyl sulfonamide compound 12 (MPT0B291, MP), or dimethyl sulfoxide (DMSO) (DM) for 4 days. After treatment, cell viability was assessed using colorimetric MTT assay. (B) In the focus formation assay, parental and TMZ-resistant (TMZ-R) U87MG cells were seeded at low density onto 60-mm plates, and treated with TMZ or MP alone or in combination at different doses every 3 days. Following a 2-week incubation period, the forming foci were stained using crystal violet. Representative images are shown. (C) TMZ-R GBM cell lines, including U87MG-R, A172-R, and P11-R cells, were treated with DMSO or different doses of MP (1, 3, 6 μM) for various time intervals (1 to 4 days). Cell viability was assessed using the MTT assay. (D) TMZ-R U87MG inoculated orthotopic mice were treated with 25 mg/kg TMZ to maintain a TMZ-resistant phenotype, and co-treated with or without 25 mg/kg MP every 2 days for 3 weeks. The brain tumors were observed using serial histology sections along the tumor using hematoxylin and eosin staining. (E) TMZ-R P3 inoculated orthotopic mice were randomly grouped and treated with DMSO, 10 mg/kg TMZ (T), or TMZ plus 10 mg/kg MP (T+M) every 2 days. Survival was plotted using a Kaplan–Meier curve. (F) Cells were transfected with HDAC1-, HDAC2-, and/or HDAC6-specific siRNAs or a nontargeting control siRNA as indicated for 2 days. After knockdown, cell viability was assessed using the MTT assay. (t-test: *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Inhibition, MTT Assay, Tube Formation Assay, Incubation, Staining, Transfection, Control, Knockdown

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: The HDAC/Sp1 pathway plays an important role in regulating cell cycle progression and proliferation. (A) Venn diagram illustrating overlaps between number of genes that were altered by more than 1.5–fold in TMZ-resistant (TMZ-R) cells and in spheroids (serum-free medium/suspension culture, S/S) following MP treatment and were also targeted by Sp1 in U87MG cells. (B) The IPA software program was applied on 139 potential HDACs/Sp1-regulated genes (the intersection genes of Sp1 ChIP-seq data and gene expression microarray data from MP-treated TMZ-R and S/S in [A]) to identify top 10 scoring canonical pathways. (C) Heat map representing the expression levels of 41 cell cycle–related genes, obtained from IPA analysis in (B), following MP treatment. (D) Relationships between MP-treated microarray data (in horizontal) and TCGA-GBM NGS data (in vertical) using Pearson’s correlation coefficient (PCC, r). Each dot represents the expression value of a cell cycle-related gene. (E) Forest plots showing hazard ratios for risk of death in low-grade and high-grade glioma patients with higher expression of the indicated gene(s). The lines on both sides denote 95% confidence intervals. All the original data (Kaplan–Meier curve) were obtained from PROGgeneV2 (Supplementary Figure 15A). Hazard ratios above 1 indicate a worse outcome. (t-test: *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Suspension, Software, ChIP-sequencing, Gene Expression, Microarray, Expressing

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: MPT0B291 induces senescence and diminished the expression stemness-related markers in both GBM spheroids and TMZ-resistant (TMZ-R) cells. (A) Mitotic P3 cells were released into the cell cycle by removing nocodazole from the culture, and then harvested at different time points as indicated. Cells were then fixed for cell cycle progression assay using flow cytometry. The percentages of cells in G1 phase and G2/M phase are shown in right panel. (B) U87MG spheroids (S/S) were treated with DMSO or MP. At 4 days posttreatment, senescence was examined using SA β-gal staining. Photomicrographs of spheroids were randomly selected in microscopic fields, and SA-β-gal positive cells were counted. (C) Cells dissociated from U87MG spheroids via trypsinization were grown in soft agar, followed by treatment with different doses of MP every 4 days. After 3 weeks of incubation at 37°C, the colonies that arose from these single cells were photographed randomly, and a histogram of average colony numbers was plotted after performing the experiment in triplicate. (D and E) Cells, as indicated, after 2 days of MP treatment were harvested, and the cell lysates were analyzed using IB with the indicated antibodies. (t-test: **P < 0.01)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Expressing, Flow Cytometry, Staining, Incubation

Journal: Neuro-Oncology

Article Title: Increased activation of HDAC1/2/6 and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma

doi: 10.1093/neuonc/noaa103

Figure Lengend Snippet: MPT0B291 decreases Sp1 binding to BMI1 and hTERT promoters. (A) The Sp1 ChIP-seq reads mapped to the promoter region of BMI1 and hTERT. Forward reads are shown in green and reverse reads are shown in red. The significance of ChIP peaks, generated using the CLC Genomics Workbench 10.1.1 software, indicated Sp1 binding loci. (B and C) U87MG spheroids were treated with DMSO or MP for 6 h, and the level of Sp1 binding to the promoter regions of BMI1 and hTERT was assessed using a ChIP assay with rabbit IgG or anti-Sp1 antibodies. DNA was then extracted from the sample for PCR with the primers as indicated. Rabbit IgG acted as a negative control for nonspecific precipitation, and E-box was used as a negative control for nonspecific binding. (D to F) U87MG spheroids were treated with different doses of MP for 2 days. After treatment, the mRNA (D) and protein (E) levels of hTERT in cells were analyzed using real-time PCR and IB, respectively. Furthermore, relative telomerase activity (F) was also detected using the TRAP assay and normalized to the value of the internal PCR control in each reaction. The cell lysis buffer was used as a negative control. The arrow points to the 36-bp internal control. (t-test: *P < 0.05, **P < 0.01)

Article Snippet: Cell Culture Human GBM cell lines U87MG and A172 (both American Type Culture Collection), 2 patient-derived GBM lines, P3 and P11, as well as their TMZ-resistant cells and tumorspheres, were cultivated in different culture media as described in previous studies.

Techniques: Binding Assay, ChIP-sequencing, Generated, Software, Negative Control, Real-time Polymerase Chain Reaction, Activity Assay, TRAP Assay, Control, Lysis

Journal: Stem cell research

Article Title: Modeling of dilated cardiomyopathy by establishment of isogenic human iPSC lines carrying phospholamban C25T (R9C) mutation (UPITTi002-A-1) using CRISPR/Cas9 editing

doi: 10.1016/j.scr.2021.102544

Figure Lengend Snippet: Characterization and validation.

Article Snippet: Pluripotency marker , TRA-1–60(S) Mouse mAb , 1:400 , Cell Signaling Technology Cat#4746S RRID: AB_2119059.

Techniques: Biomarker Discovery, Immunofluorescence, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Gene Expression, Microarray, Sequencing, Plasmid Preparation, Modification, Mutagenesis, CRISPR, In Vitro

Journal: Stem cell research

Article Title: Modeling of dilated cardiomyopathy by establishment of isogenic human iPSC lines carrying phospholamban C25T (R9C) mutation (UPITTi002-A-1) using CRISPR/Cas9 editing

doi: 10.1016/j.scr.2021.102544

Figure Lengend Snippet: Reagents details.

Article Snippet: Pluripotency marker , TRA-1–60(S) Mouse mAb , 1:400 , Cell Signaling Technology Cat#4746S RRID: AB_2119059.

Techniques: Immunocytochemistry, Marker, Staining, Mutagenesis, DNA Amplification, DNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, CRISPR, Plasmid Preparation, Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Primer sequences for the genes to validate the microarray analysis by RT-PCR.

Article Snippet: Primary antibodies used included rabbit anti-Tollip monoclonal antibody (1:400), rabbit anti-NF-κB monoclonal antibody (1:400), rabbit anti-IRAK1 monoclonal antibody (1:400), rabbit anti-TLR4 monoclonal antibody (1:400), rabbit anti-TRAF6 monoclonal antibody (1:400), rabbit anti-VEGF-α monoclonal antibody (1:400), rabbit anti-Nrf2 monoclonal antibody (1:400), mouse anti-GAPDH monoclonal antibody (1:400) were purchased from Santa Cruz Biotechnology, Inc..

Techniques: Microarray

Journal: International Journal of Molecular Medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Effect of XBJ on the mRNA expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue. Groups of mice were challenged with CLP and treated with XBJ 24 h later. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Representative RT-PCR shows the level of Tollip, IRAK1, TLR4, NF-κB65, and TRAF6 expression in the four rat groups. M, marker; A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Primary antibodies used included rabbit anti-Tollip monoclonal antibody (1:400), rabbit anti-NF-κB monoclonal antibody (1:400), rabbit anti-IRAK1 monoclonal antibody (1:400), rabbit anti-TLR4 monoclonal antibody (1:400), rabbit anti-TRAF6 monoclonal antibody (1:400), rabbit anti-VEGF-α monoclonal antibody (1:400), rabbit anti-Nrf2 monoclonal antibody (1:400), mouse anti-GAPDH monoclonal antibody (1:400) were purchased from Santa Cruz Biotechnology, Inc..

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Control

Journal: International Journal of Molecular Medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Administration of XBJ enhanced the expression of Tollip protein protein, and inhibition TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Statistical summary of the densitometric analysis of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ** P<0.01 vs. the normal control group and sham operation group. # P<0.05, ## P<0.01 vs. the control group.

Article Snippet: Primary antibodies used included rabbit anti-Tollip monoclonal antibody (1:400), rabbit anti-NF-κB monoclonal antibody (1:400), rabbit anti-IRAK1 monoclonal antibody (1:400), rabbit anti-TLR4 monoclonal antibody (1:400), rabbit anti-TRAF6 monoclonal antibody (1:400), rabbit anti-VEGF-α monoclonal antibody (1:400), rabbit anti-Nrf2 monoclonal antibody (1:400), mouse anti-GAPDH monoclonal antibody (1:400) were purchased from Santa Cruz Biotechnology, Inc..

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Control

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: (a) TRAF6 mRNA in CD34 + BM cells from low-risk MDS patients (n = 36) and age-matched healthy controls (HC; n = 7) (left), and LSK cells from Vav -TRAF6 (n = 6) and WT FVB/NJ (n = 4) mice (primers are designed to measure both endogenous mouse Traf6 and ectopic human TRAF6). P = 0.02. ( b ) Disease-free survival of Vav -TRAF6 (n = 60) and WT FVB/NJ mice (n = 40) *, P = 0.009. ( c ) Complete blood counts for WT FVB/NJ (n = 33) and moribund Vav -TRAF6 (n = 18) mice. *. P < 0.01. ( d ) BM neutrophil dysplasia in age-matched WT (n = 3) and Vav -TRAF6 (n = 5) FVB/NJ mice. At least 100 cells were examined for each mouse. *, P = 0.04. ( e ) BM mononuclear cell counts from WT (n = 3) and Vav -TRAF6 FVB/NJ mice (n = 4). *, P = 0.004. ( f ) Representative Wright-Giemsa-stained peripheral blood smears (PB) and bone marrow cytospins (BM) from WT (FVB/NJ) and 2 moribund Vav -TRAF6 mice. The red arrow indicates dysplastic myeloid cells with Pseudo-Pelger Hüet anomaly. Scale bars, 10 µm (top) and 7.5 µm (bottom) ( g ) Number of long-term hematopoietic stem cells (LT-HSC, P = 0.0056), short-term HSC (ST-HSC, P = 0.0011), multipotent progenitors (MPP, P = 0.014), common myeloid progenitors (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-monocytic progenitor (GMP) in the BM of WT (C57Bl/6) (n = 4) and Vav -TRAF6 (n = 4) ~6 month old mice. ( h ) Colony numbers were determined after primary and secondary plating LSK BM cells from WT (FVB/NJ; n = 3) and Vav -TRAF6 (n = 3) mice in methylcellulose. *, P < 0.001. ( i ) FACS analysis of WT (C57Bl/6) or Vav -TRAF6 donor-derived (CD45.2 + ) and competitor-derived (CD45.1 + ) mononuclear cells from peripheral blood 12 weeks post competitive transplantation (left panel). Proportion of myeloid (CD11b + ) and lymphoid (CD3 + and B220 + ) cells within the donor-derived compartment (gated on CD45.2 + ) after competitive transplantation with either BM mononuclear cells (MNC, left) or LT-HSC (right). (n = 8 mice per genotype) (right panel). *, P < 0.05. ( j ) Colony formation in methylcellulose after serial replating of GFP-sorted Lin − BM cells (C57Bl/6) transduced with retroviral vectors (MSCV-IRES-GFP) encoding WT (n = 3) or an E3 ligase-mutant TRAF6 (TRAF6 C70A , n = 3). *, P < 0.01. ( k ) Proportion of donor-derived (gated on CD45.2 + ;GFP + ) peripheral blood myeloid (Cd11b + ) and lymphoid (CD3 + and B220 + ) cells were examined by FACS for competitively transplanted GFP-sorted Lin − BM cells (C57Bl/6) transduced with retroviral vectors encoding WT (n = 5) or an E3 ligase-mutant TRAF6 (TRAF6 C70A , n = 10) co-expressing GFP (MSCV-IRES-GFP). *, P < 0.05. Data are from three experiments ( a, c–g ; means and s.e.m.) or from two experiments ( h–k ; means and s.e.m.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: Staining, Derivative Assay, Transplantation Assay, Transduction, Retroviral, Mutagenesis, Expressing

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: ( a ) In vitro Ub reconstitution assays were performed on the identified candidates from the cell-based screen with purified TRAF6 (n = 29). Shown is the substrate ubiquitination in the presence of TRAF6 (F532 CyDye) as compared to without TRAF6 (B532 CyDye) for 2 independent replicates. ( b ) Exon-level microarray expression analysis of LSK from 6 month WT (FVB/NJ, n = 4) and Vav -TRAF6 mice (n = 3). ( c ) Motif enrichment in differentially spliced exons from Vav -TRAF6 LSK as compared to WT LSK in the form of position frequency matrix motifs from the CisBP-RNA database. Each point represents one motif. Motifs are sorted left to right in decreasing order of significance. Motifs enriched in Vav -TRAF6 LSK are above the horizontal dotted line. hnRNPA1, P = 9.7x10 −5 . ( d ) Enrichment scores for exon usage patterns for Vav -TRAF6 LSK as compared to 46 known splicing factor exon usage patterns. Each point represents the enrichment Z-score for an individual splicing pattern of the splicing factor patterns examined. Exon usage patterns enriched in Vav -TRAF6 LSK are above the horizontal dotted line. hnRNPA1, P = 0.017. Data are from two experiments ( a ; means and s.d.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: In Vitro, Purification, Ubiquitin Proteomics, Microarray, Expressing

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: ( a ) In vitro ubiquitin reconstitution assay performed in duplicate and analyzed by CyeDye fluorescence (F532 subtracted from background, B532) for hnRNPA1, TAK1, and Tubulin (TUBB; a negative control). Individual assays contained recombinant Ub, UBE1, Ubc13/UBE2V1, ATP, and the indicated substrates. nd, not detectable. ( b ) HEK293 cells transfected with MYC-hnRNPA1, FLAG-TRAF6, and either WT HA-Ub or mutant Ub that form only K63- (Ub K63 ) or K48- (Ub K48 ) linkages were immunoblotted for HA (Ub) on IP MYC-hnRNPA1. ( c ) HEK293 cells transfected with MYC-hnRNPA1, and either WT or E3 ligase defective (C70A) TRAF6 were IB for IP MYC-hnRNPA1 (anti HA-Ub). ( d ) Schematic representation of hnRNPA1 protein, and corresponding lysine-to-arginine mutations within the RNA recognition motif (RRM) (above image). HEK293 cells transfected with WT, Lys3-mutant hnRNPA1 (K3R), or hnRNPA1 with mutation of the lysines within the first RRM domain (K–R) were immunoprecipitated (IP) for MYC-hnRNPA1 and then immunoblotted for HA (Ub). Data are from two experiments ( a ; means and s.d.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: In Vitro, Ubiquitin Proteomics, Reconstitution Assay, Fluorescence, Negative Control, Recombinant, Transfection, Mutagenesis, Immunoprecipitation

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: ( a ) Immunoprecipitation and immunobloting of ubiquitinated hnRNPA1 from WT (FVB/NJ) and Vav -TRAF6 Lin − BM cells and LPS-treated WT (C57Bl/6) ( Traf6 +/+ ) and TRAF6-deficient ( Traf6 −/− ) Lin − BM cells. ( b ) hnRNPA1 mRNA and protein expression was determined following expression of shRNAs targeting hnRNPA1 (shA1) or non-targeting shRNA (shCtl). ( c ) Colony formation in methylcellulose after serial replating of GFP-sorted WT (FVB/NJ, n = 3) and Vav -TRAF6 (n = 3) Lin − BM cells transduced with shRNAs targeting hnRNPA1 (shA1) or control shRNA (shCtl) co-expressing GFP. *, P < 0.05; # , P = 0.08. ( d ) Chimerism of donor-derived (CD45.2 + GFP + ) peripheral blood cells from lethally-irradiated recipient mice receiving Lin − BM cells transduced with shRNAs targeting hnRNPA1 (shA1) or control shRNA (shCtl) co-expressing GFP competitively transplanted with wild-type (CD45.1 + ) total BM cells (n = 8 per genotype). *, P < 0.05. Data are from two experiments ( b–d ; means and s.e.m.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: Immunoprecipitation, Western Blot, Expressing, shRNA, Transduction, Control, Derivative Assay, Irradiation

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: ( a ) Significantly excluded or included exons as determined by FIRMA index (−4.0 ≥ a ≤ 4.0) in Vav -TRAF6 versus WT (FVB/NJ) LSK (MIDAS P < 0.05). Each point represents a differentially spliced exon. Highlighted are the top 3 differentially spliced exons in Vav -TRAF6 LSK. ( b ) Schematic representation of the mouse Arhgap1 gene structure with the approximate position of exons and introns. Overview of Arhgap1 exon usage is shown for WT (FVB/NJ) (black lines) and Vav -TRAF6 LSK (red lines). The average of FIRMA index values for individual exons of Arhgap1 in WT (n = 4) and Vav -TRAF6 LSK (n = 3) are shown below. FIRMA index scores indicate exon exclusion (negative values) or exon retention (~0). ( c ) RT-PCR analysis (using primers flanking exon 2) of the Arhgap1 cassette exon in Vav -TRAF6 and WT (FVB/NJ) LSK (left), and in LPS-treated WT Lin − BM cells (right). Values under the plot represent the short isoform as a percentage of the short and long isoforms. (d) Sequence analysis of the Arhgap1 cassette exon using primers flanking skipped exon 2 isolated from Vav -TRAF6 LSK. ( e ) RT-PCR analysis (using primers flanking exon 2, as depicted in the schematic) of the Arhgap1 cassette exon in WT (FVB/NJ) or Vav -TRAF6 Lin − BM cells were transduced with non-targeting shRNA (shCtl) or shRNA targeting mouse hnRNPA1 (shA1). Summary of Arhgap1 exon 2 exclusion calculated based on the short isoform as a percentage of the short and long isoforms is shown above. n = 3 per group; # , P = 0.06; *, P = 0.04. ( f ) RT-PCR analysis (using primers flanking exon 2, as depicted in the schematic) of the Arhgap1 cassette exon in WT (C57Bl/6) ( Traf6 +/+ ;Mx1Cre) and TRAF6-deficient Lin − BM cells ( Traf6 fl/fl ;Mx1Cre mice treated with PolyIC) stimulated with 10 ng/mL of LPS for 2 hrs. Summary of Arhgap1 exon 2 exclusion calculated based on the short isoform as a percentage of the short and long isoforms is shown above. n = 3 per group; *, P < 0.05. ( g ) RT-PCR analysis of a minigene plasmid containing the Arhgap1 cassette exon 2 with 50 bp of flanking intron sequences (using primers flanking exon 2, as depicted in the schematic on the left) in HEK293 cells (HEK293- Arhgap1 exon2) transfected with hnRNPA1. Summary of Arhgap1 exon 2 exclusion from the minigene calculated based on the short isoform as a percentage of the short and long isoforms is shown above from 2 independent experiments. *, P = 0.02. ( h ) RT-PCR analysis of a minigene plasmid containing the Arhgap1 cassette exon 2 (using primers flanking exon 2, as depicted in the schematic on the left) in HEK293- Arhgap1 exon2 cells transfected with WT (n = 7) and an E3 ligase-mutant (C70A; n = 5) TRAF6. Summary of Arhgap1 exon 2 exclusion from the minigene was calculated based on the short isoform as a percentage of the short and long isoforms is shown above from 2 independent experiments. *, P = 0.03. ( i ) RT-PCR analysis of a minigene plasmid containing the Arhgap1 cassette exon 2 (using primers flanking exon 2, as depicted in the schematic on the left) in HEK293- Arhgap1 exon2 cells transfected with TRAF6 and hnRNPA1 (WT and lysine mutants). Summary of Arhgap1 exon 2 exclusion from the minigene was calculated based on the short isoform as a percentage of the short and long isoforms is shown above from 2 independent experiments (n = 2 per group). Vec, vector; A1 WT , hnRNPA1 WT; A1 K3R , hnRNPA1 with K3R mutation; A1 K-R , hnRNPA1 with mutation of all lysines within the first RNA recognition motif. *, P < 0.01; **, P = 0.003. ( j ) qRT-PCR of Arhgap1 cassette exon 2 expressed from the minigene plasmid following RNA immunoprecipitation of Myc-hnRNPA1 in HEK293- Arhgap1 exon2 cells transfected with Myc-hnRNPA1 (WT and K-R) and TRAF6. Shown is the average of 2 independent experiments (n = 2 per group). *, P < 0.01. Data are from two experiments ( e–j ; means and s.e.m.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Isolation, Transduction, shRNA, Plasmid Preparation, Transfection, Mutagenesis, Quantitative RT-PCR, RNA Immunoprecipitation

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: ( a ) Schematic of the Arhgap1 5’-UTR and coding region. uORF, upstream open reading frame; HA, hemagglutinin. The indicated Arhgap1 5’-UTR cDNA fusions were transfected (pCMV-GFP) into HEK293 cells and evaluated by Arhgap1 and HA immunoblotting. Transfected Arhgap1 5’-UTR cDNA were measured by RT-PCR using primers aligned to Arhgap1 coding sequences. ( b ) Microarray and qRT-PCR analysis of Arhgap1 total RNA from LSK of WT (FVB/NJ) and Vav -TRAF6 mice. ( c ) Immunoblotting of Arhgap1 in Lin − BM cells from WT (FVB/NJ) and Vav -TRAF6 mice. Cdc42 activity (Cdc42-GTP) in WT (FVB/NJ) and Vav -TRAF6 Lin − BM cells was determined by a pull-down/immunoblot assay. Shown is a representative blot from one WT (FVB/NJ) and three Vav -TRAF6 mice. ( d) Densitometric analysis of Arhgap1 protein and Cdc42-GTP (WT, n = 3; Vav -TRAF6, n = 5). *, P < 0.05. Data are from three experiments ( b,d ; means and s.e.m.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Microarray, Quantitative RT-PCR, Activity Assay

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: (a) FACS analysis of donor-derived (gated on CD45.2 + ) myeloid (CD11b + Gr1 + ) and lymphoid (B220 + and CD3 + ) proportions 8 weeks post transplantation for competitively transplanted WT (C57Bl/6) and Vav -TRAF6 LT-HSC, and for Vav -TRAF6 LT-HSC treated in vitro with CASIN (or DMSO) for 24 hr. *, P < 0.05. ( b ) Summary of independent experiments of myeloid and lymphoid proportions of donor-derived (gated on CD45.2 + ) peripheral blood shown as a ratio (CD11b/[B220 + CD3]). ( c ) Peripheral blood myeloid (CD11b + ) and lymphoid (B220 + and CD3 + ) chimerism (CD45.2 + ) of donor-derived cells determined 8 weeks after competitive transplantation with Vav -TRAF6 LT-HSC treated with CASIN. (n = 6 per group). *, P < 0.005. (d) Donor-derived LT-HSC cells (CD45.2 + ) from Vav -TRAF6 mice and competitor BM cells (CD45.1 + ) were transplanted into lethally-irradiated recipient mice (CD45.1 + ). Three month post secondary transplantation, the recipients were treated with 30 mg/kg CASIN and vehicle control (PBS) for 16 days. Ratio of donor-derived (gated on CD45.2 + ) and competitor-derived (gated on CD45.1 + ) myeloid (CD11b) and lymphoid (CD3 and B220) cells was determined by flow cytometry 4 weeks post-CASIN treatment in vivo. Donor-derived chimerism is normalized (1.0) for each mouse at week 1 (n = 5 per group). *, P = 0.007. Data are from two experiments ( b–d ; means and s.e.m.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: Derivative Assay, Transplantation Assay, In Vitro, Irradiation, Control, Flow Cytometry, In Vivo

Journal: Nature immunology

Article Title: Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia

doi: 10.1038/ni.3654

Figure Lengend Snippet: ( a ) RT-PCR using primers flanking endogenous ARHGAP17 exon 18 or exons 17 and 18 (as depicted in the schematic on the left) in HEK293 or MDSL cells expressing shRNAs targeting TRAF6 (shTRAF6) or a non-targeting control (shCtl). ( b ) Immunoblotting of ARHGAP17 in independently transduced MDSL cells (MDSL-1-2) expressing a non-targeting shRNA (shCtl) or shRNA targeting TRAF6 (shTRAF6). ( c ) Sequence analysis of the ARHGAP17 exons 16, 17, 18, and 19 in MDSL cells. ( d ) RT-PCR using primers flanking endogenous ARHGAP17 exon 18 (as depicted in the schematic on the left) in MDS patient BM mononuclear cells with high (>2-fold TRAF6 , n = 3) or low (< 1.0-fold TRAF6 , n =3) TRAF6 mRNA, and 1 normal BM sample. ( e ) qRT-PCR of ARHGAP17 exon 18 using primers to the exon 17–19 junction and normalized to total ARHGAP17 in MDS/AML BM cells (n = 35). Spearman correlation: r = 0.3854, P = 0.014. ( f ) Cdc42-GTP pulldown assays in MDSL cells expressing non-targeting shRNA (shCtl) or shRNA targeting TRAF6 (shTRAF6), or in MDSL treated with 2.5 µM CASIN. ( g ) Cdc42-GTP pulldown assays in MDSL expressing non-targeting shRNA (shCtl) or shRNA targeting ARHGAP17 (shGAP17). ( h ) Colony formation in methylcellulose of human CD34 + cells expressing a non-targeting shRNA (shCtl) or shRNA targeting ARHGAP17 (shGAP17) (n = 3). *, P = 0.047. Data are from three experiments ( h ; means and s.e.m.).

Article Snippet: Densitometry was determined with Image J. Quantitative PCR was performed with Taqman Master Mix (Life Technologies) for human TRAF6 (Cat# 4331182, Assay# Hs00371512_g1), human hnRNPA1 (Cat# 4453320, Assay# Hs01656228_g1), mouse hnRNPA1 (Cat# 4448892, Assay# Mm02528230_g1), human GAPDH (Cat# 4331182, Assay# Hs02758991_g1), mouse GAPDH (Cat# 4453320, Assay# Mm99999915_g1) on an Applied Biosystems StepOne Plus Real-Time PCR System.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot, shRNA, Sequencing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Kaplan-Meier plot showing overall survival of bladder cancer patients with differential TRAF4 expression, data obtained and reproduced from TCGA (The Human Protein Atlas), the median FKPM value was taken as TRAF4 expression cut-off. B Graph representing TRAF4 expression through scores obtained from immunohistochemistry of tissue microarray, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test; n.s. indicates non-significant P value. C Representative images of immunohistochemistry performed for TRAF4 expression on tissue microarray from stages 1-3 of bladder tumours. D Violin plot shows TRAF4 expression levels (and distribution) across different subtypes of bladder cancer, Her2L: Her2-like (n=253), Pap: Papillary (n=674), Lum: Luminal (n=107), Neu: Neural (n=448), SCC: Squamous cell carcinoma (n=333) and Mes: Mesenchymal (n=308). Black bars in the middle of the distribution represents Median. The subtypes have been arranged according to their EMT scores. E Plot represents EMT scores in 59 bladder cancer cell lines, green bars indicate cell lines with negative EMT scores, red bars indicate cell lines with positive EMT scores; red arrowheads indicate cell lines taken for further investigation. F Regression plot for TRAF4 expression levels vs EMT scores in 59 bladder cancer cell lines. G Immunoblot analysis showing TRAF4 and other EMT markers protein expression. H Real-time PCR showing TRAF4 mRNA expression in cell lines. Error bars represent ± SD. Epithelial cell lines (green bars) have significantly higher TRAF4 expression than mesenchymal cell lines (red bars), ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test.

Article Snippet: The following vectors and its derivates were used: pcDNA3.1 (6xMyc) TRAF4, pFLAG-CMV SMURF1, GFP-ERK1 was a gift from Rony Seger (Addgene plasmid # 14747)[ ] and GFP-TRAF4 was a gift from Ying Zhang (Addgene plasmid # 58318)[ ].

Techniques: Expressing, Immunohistochemistry, Microarray, Two Tailed Test, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Real-time PCR result showing changes in TRAF4 mRNA levels in mesenchymal cell lines after treatment with 5’-Azacitydine; error bars represent ± SD, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. B Real-time PCR result showing changes in CDH1 mRNA levels in cell lines after treatment with 5’-Azacitydine; error bars represent ± SD, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. C Immunoblot results showing endogenous TRAF4 levels after treatment with cycloheximide (CHX) using the indicated cell lines. D Schematic representation of TRAF4 showing distinct domain structures and candidate phosphorylated serine and threonine sites derived from mass spectrometric analysis. E Immunoblot result from 293T transfected with expression constructs for either TRAF4 or TRAF4 glutamic acid mutants. F Immunoblot result from 293T transfected with expression constructs for either TRAF4 or TRAF4 alanine mutants. G Immunoprecipitation result from 293T transfected with the indicated plasmids and immunoprecipitated with Myc antibody. H Immunofluorescence images of HT1376 transfected with expression constructs for either GFP-TRAF4 or GFP-S334 mutants, red arrowheads indicate localization of GFP-TRAF4 and GFP-S334A at the plasma membrane. I Representative structures from molecular dynamics simulations of the TRAF domain trimer from TRAF4 in complex with PIP3-dic4 lipid molecules in the unphosphorylated and phosphorylated states of S334. The TRAF domain is shown in electrostatic surface representation (created using the APBS plugin through the Pymol molecular visualization software, Schrondinger) and the color gradient from blue to red indicates the range of electrostatic surface potential kT/e values from strongly positive (+3.0) to strongly negative (−3.0). The residues that are involved in specifically interacting with the lipid headgroup along with the lipid are explicity shown in stick representation and labelled. The lower graphs indicates the binding energies of the individual lipids for the respective systems; the energies were computed using the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method by following the same procedure and parameters as described previously.

Article Snippet: The following vectors and its derivates were used: pcDNA3.1 (6xMyc) TRAF4, pFLAG-CMV SMURF1, GFP-ERK1 was a gift from Rony Seger (Addgene plasmid # 14747)[ ] and GFP-TRAF4 was a gift from Ying Zhang (Addgene plasmid # 58318)[ ].

Techniques: Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, Derivative Assay, Transfection, Expressing, Construct, Immunoprecipitation, Immunofluorescence, Clinical Proteomics, Membrane, Software, Binding Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Real-time PCR result from RT4 cells showing mRNA expression levels of genes indicated; error bars represent ± SD, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. B Immunoblot result showing EMT markers protein expression changes in RT4 cells upon TRAF4 knockdown. C RT4 cell colonies imaged by brightfield (top panels) or after exposure to CellMask™ Orange plasma membrane stain (bottom panels), scale: 25μM. D Images depicting RT4 spheroids made from control (empty vector pLKO) and TRAF4 knockdown cells (sh5), scale: 200μM. The graph represents circularities calculated from five independent spheroids of different sizes. Error bars represent ± SD, ** P ≤ 0.01 calculated using two-tailed student’s t test. E Real-time PCR result from HT1376 cells showing mRNA expression of the genes indicated; error bars represent ± SD, * P ≤ 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. F Immunoblot result showing EMT markers protein expression changes in HT1376 cells upon TRAF4 knockdown. G Representative images of transwell assays performed on HT1376 cells, cells were stained with crystal violet, scale: 200μM. H Quantification of number of migrated cells from four random fields; error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test.

Article Snippet: The following vectors and its derivates were used: pcDNA3.1 (6xMyc) TRAF4, pFLAG-CMV SMURF1, GFP-ERK1 was a gift from Rony Seger (Addgene plasmid # 14747)[ ] and GFP-TRAF4 was a gift from Ying Zhang (Addgene plasmid # 58318)[ ].

Techniques: Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test, Western Blot, Knockdown, Clinical Proteomics, Membrane, Staining, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Immunoblot result of RT4 control (empty vector pLKO) and TRAF4 knockdown cells (sh4 and sh5) probed with the indicated antibodies. B Immunoblot result of HT1376 control (empty vector pLKO) and TRAF4 knockdown cells (sh4) probed with the indicated antibodies. Numbers represent relative quantification of SMURF1 levels with respect to GADPH. C Real-time PCR result showing SMURF1 mRNA expression levels in RT4 and HT1376 (control and TRAF4 knockdown) cells; error bars represent ± SD, n.s. indicates non-significant P value. D Immunoblot result from 293T cells transfected with the indicated plasmids. E Ubiquitination assay performed in 293T cells with Flag antibodies, over-expressing the indicated plasmids. Cells were treated with MG132 (2μM) overnight prior to lysis. Representative result from three independent experiments. F Real-time PCR showing Smurf1 mRNA levels in MBT-2 control (pLKO vector) and Smurf1 knockdown cells (sh1 and sh2); error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test. G Representative images of transwell assay performed on MBT-2 control and Smurf1 knockdown cells, which were stained with crystal violet, scale: 200μM. H Quantification of number of migrated cells from four random fields, error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test. I Graph showing relative wound density from Incucyte, images were obtained every 1 hour after wound was produced, T24 cells were treated with either DMSO or SMURF1 inhibitor, A01 (10μM); error bars represent ± SEM, *** P ≤ 0.001 calculated using two-tailed student’s t test. Representative result from three independent experiments . J Representative images from graph in I, brown area represents the cell coverage and grey area is the wound produced and remaining. K MTS assay performed on T24, cells were treated with either control (DMSO) or SMURF1i, A01 at 5μM. Absorbance was read at the indicated time points; error bars represent ± SD from three sample replicates, * P ≤ 0.05 calculated using two-tailed student’s t test.

Article Snippet: The following vectors and its derivates were used: pcDNA3.1 (6xMyc) TRAF4, pFLAG-CMV SMURF1, GFP-ERK1 was a gift from Rony Seger (Addgene plasmid # 14747)[ ] and GFP-TRAF4 was a gift from Ying Zhang (Addgene plasmid # 58318)[ ].

Techniques: Western Blot, Control, Plasmid Preparation, Knockdown, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing, Transfection, Ubiquitin Proteomics, Lysis, Two Tailed Test, Transwell Assay, Staining, Produced, MTS Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Immunoblot showing T24 cells stably expressing either control vector (myc-tag), TRAF4 or catalytically inactive TRAF4 mutant (C/A: cysteine to alanine at residue C18). B Representative images of transwell assays performed on T24 cells, cells were stained with crystal violet, scale: 100μM. C Quantification of number of migrated cells from four random fields, error bars represent ± SD, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. D Graph showing relative wound density result from Incucyte. Representative result from three independent experiments; error bars represent ± SEM, *** P ≤ 0.001 calculated using two-tailed student’s t test. E Representative images from graph in D, brown area represents the cell coverage and grey area is the wound produced and remaining. F MTS assay performed with either control or TRAF4 stably expressing T24 cells. Absorbance was read at the indicated time points; error bars represent ± SD from three sample replicates, *** P ≤ 0.001 calculated using two-tailed student’s t test. G Immunoblot result showing MBT-2 cells stably expressing either control (empty vector with Myc-tag) or Myc-TRAF4. H Representative images of transwell assays performed on MBT-2 control and TRAF4 over-expressing cells, which were stained with crystal violet, scale: 200μM. I Quantification of number of migrated cells from four random fields, error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test.

Article Snippet: The following vectors and its derivates were used: pcDNA3.1 (6xMyc) TRAF4, pFLAG-CMV SMURF1, GFP-ERK1 was a gift from Rony Seger (Addgene plasmid # 14747)[ ] and GFP-TRAF4 was a gift from Ying Zhang (Addgene plasmid # 58318)[ ].

Techniques: Western Blot, Stable Transfection, Expressing, Control, Plasmid Preparation, Mutagenesis, Residue, Staining, Two Tailed Test, Produced, MTS Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Graph showing differences in enrichment scores when TRAF4 is over-expressed in T24 cells. Gene signatures of eleven major cancer associated signalling pathways were considered for analysis. B Venn diagram showing number of genes which are up-regulated in two independent hairpins targeting TRAF4 in HT1376 (pink) and down-regulated with stable over-expression of TRAF4 in T24 (green) compared to their respective controls. 252 genes in the middle represents the reciprocally affected common genes. Results were obtained from four independent replicates for each sample. C Venn diagram showing number of genes which are down-regulated in two independent hairpins targeting TRAF4 in HT1376 (green) and up-regulated with stable over-expression of TRAF4 in T24 (pink) compared to their respective controls. 96 genes in the middle represents the reciprocally affected common genes. Results were obtained from four independent replicates for each sample. D Heatmap showing the common mis-regulated genes within the BMP, NF-κB and EMT gene signatures in both cell lines. E Real-time PCR result showing mRNA expression levels of the indicated genes in control (empty vector myc-tag) vs TRAF4 over-expressing T24 cells upon stimulation of BMP6 (50ng/ml) for 1 hour, error bars represent ± SD, * P ≤ 0.05, ** P ≤ 0.01, calculated using two-tailed student’s t test. F Real-time PCR showing mRNA expression levels of ID1, ID2 and ID3 in the indicated cell lines. G Graph showing relative wound density result from Incucyte, images were obtained every 1 hour after wound was produced, T24 cells were treated with BMP6 (50ng/ml); error bars represent ± SEM, *** P ≤ 0.001 calculated using two-tailed student’s t test. H Representative images from graph in G, brown area represents the cell coverage and grey area is the wound produced and remaining. I MTS assay performed with T24 cells, either control and stimulated with BMP6 (50ng/ml). Absorbance was read at the indicated time points; error bars represent ± SD from three sample replicates, ** P ≤ 0.01 calculated using two-tailed student’s t test.

Article Snippet: The following vectors and its derivates were used: pcDNA3.1 (6xMyc) TRAF4, pFLAG-CMV SMURF1, GFP-ERK1 was a gift from Rony Seger (Addgene plasmid # 14747)[ ] and GFP-TRAF4 was a gift from Ying Zhang (Addgene plasmid # 58318)[ ].

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Control, Plasmid Preparation, Two Tailed Test, Produced, MTS Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Luciferase reporter assay in 293T transfected with BRE-luciferase reporter, SV40 Renilla and either empty vector control or TRAF4, transfected cells were stimulated overnight with BMP6 (50ng/ml) and/or TNF-α (10ng/ml) where indicated, error bars represent ± SD, * P ≤ 0.05, *** P ≤ 0.001, calculated using two-tailed student’s t test, n.s. indicates non-significant P value. Representative result from three independent experiments. B Real-time PCR result showing mRNA expression levels of the indicated genes in control (empty vector myc-tag) vs TRAF4 over-expressing T24 cells upon stimulation of BMP6 (50ng/ml) and/or TNF-α (10ng/ml) where indicated for 1 hour. Error bars represent ± SD, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, calculated using two-tailed student’s t test. C Regression analysis showing correlation between TRAF4 expression levels (scores) and pSMAD1/5/8 scores from bladder cancer patients. Pearson’s test was used to determine the correlation between TRAF4 and pSMAD1/5/8 scores. D Regression analysis showing correlation between TRAF4 expression levels (scores) and p-p65 scores from bladder cancer patients. Pearson’s test was used to determine the correlation between TRAF4 and p-p65 scores. E Representative images of continuous sections of tissue microarray samples probed with the indicated antibodies using fluorescent immunohistochemistry. The magnified insets for pSMAD1/5/8 shows nuclear staining. F Schematic representation of TRAF4 signalling dynamics between epithelial and mesenchymal bladder cancer cells. Ub denotes Ubiquitin, Me is methylated promoter DNA and P stands for Phosphorylation of S334 site.

Article Snippet: The following vectors and its derivates were used: pcDNA3.1 (6xMyc) TRAF4, pFLAG-CMV SMURF1, GFP-ERK1 was a gift from Rony Seger (Addgene plasmid # 14747)[ ] and GFP-TRAF4 was a gift from Ying Zhang (Addgene plasmid # 58318)[ ].

Techniques: Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Control, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Microarray, Immunohistochemistry, Staining, Ubiquitin Proteomics, Methylation, Phospho-proteomics

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Kaplan-Meier plot showing overall survival of bladder cancer patients with differential TRAF4 expression, data obtained and reproduced from TCGA (The Human Protein Atlas), the median FKPM value was taken as TRAF4 expression cut-off. B Graph representing TRAF4 expression through scores obtained from immunohistochemistry of tissue microarray, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test; n.s. indicates non-significant P value. C Representative images of immunohistochemistry performed for TRAF4 expression on tissue microarray from stages 1-3 of bladder tumours. D Violin plot shows TRAF4 expression levels (and distribution) across different subtypes of bladder cancer, Her2L: Her2-like (n=253), Pap: Papillary (n=674), Lum: Luminal (n=107), Neu: Neural (n=448), SCC: Squamous cell carcinoma (n=333) and Mes: Mesenchymal (n=308). Black bars in the middle of the distribution represents Median. The subtypes have been arranged according to their EMT scores. E Plot represents EMT scores in 59 bladder cancer cell lines, green bars indicate cell lines with negative EMT scores, red bars indicate cell lines with positive EMT scores; red arrowheads indicate cell lines taken for further investigation. F Regression plot for TRAF4 expression levels vs EMT scores in 59 bladder cancer cell lines. G Immunoblot analysis showing TRAF4 and other EMT markers protein expression. H Real-time PCR showing TRAF4 mRNA expression in cell lines. Error bars represent ± SD. Epithelial cell lines (green bars) have significantly higher TRAF4 expression than mesenchymal cell lines (red bars), ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test.

Article Snippet: The following antibodies were used: TRAF4 1:2000 (D1N3A, CST), E-cadherin 1:1000 (Cat no. 610181, BD), N-cadherin 1:1000 (Cat no. 610920, BD), Vimentin 1:5000 (CST), SLUG 1:1000 (C19G7, CST), SNAIL 1:1000 (C15D3, CST), Flag 1:5000 (M2, Sigma Aldrich), phospho-Serine 1:1000 (612546, BD), SMURF1 1:1000 (45-K, Santa Cruz), Myc 1:5000 (9E10, Santa Cruz), HA 1:5000 (Y11, Santa Cruz), GFP 1:5000 (FL, Santa Cruz) and GAPDH 1:10,000 (MAB374, Millipore).

Techniques: Expressing, Immunohistochemistry, Microarray, Two Tailed Test, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Real-time PCR result showing changes in TRAF4 mRNA levels in mesenchymal cell lines after treatment with 5’-Azacitydine; error bars represent ± SD, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. B Real-time PCR result showing changes in CDH1 mRNA levels in cell lines after treatment with 5’-Azacitydine; error bars represent ± SD, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. C Immunoblot results showing endogenous TRAF4 levels after treatment with cycloheximide (CHX) using the indicated cell lines. D Schematic representation of TRAF4 showing distinct domain structures and candidate phosphorylated serine and threonine sites derived from mass spectrometric analysis. E Immunoblot result from 293T transfected with expression constructs for either TRAF4 or TRAF4 glutamic acid mutants. F Immunoblot result from 293T transfected with expression constructs for either TRAF4 or TRAF4 alanine mutants. G Immunoprecipitation result from 293T transfected with the indicated plasmids and immunoprecipitated with Myc antibody. H Immunofluorescence images of HT1376 transfected with expression constructs for either GFP-TRAF4 or GFP-S334 mutants, red arrowheads indicate localization of GFP-TRAF4 and GFP-S334A at the plasma membrane. I Representative structures from molecular dynamics simulations of the TRAF domain trimer from TRAF4 in complex with PIP3-dic4 lipid molecules in the unphosphorylated and phosphorylated states of S334. The TRAF domain is shown in electrostatic surface representation (created using the APBS plugin through the Pymol molecular visualization software, Schrondinger) and the color gradient from blue to red indicates the range of electrostatic surface potential kT/e values from strongly positive (+3.0) to strongly negative (−3.0). The residues that are involved in specifically interacting with the lipid headgroup along with the lipid are explicity shown in stick representation and labelled. The lower graphs indicates the binding energies of the individual lipids for the respective systems; the energies were computed using the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method by following the same procedure and parameters as described previously.

Article Snippet: The following antibodies were used: TRAF4 1:2000 (D1N3A, CST), E-cadherin 1:1000 (Cat no. 610181, BD), N-cadherin 1:1000 (Cat no. 610920, BD), Vimentin 1:5000 (CST), SLUG 1:1000 (C19G7, CST), SNAIL 1:1000 (C15D3, CST), Flag 1:5000 (M2, Sigma Aldrich), phospho-Serine 1:1000 (612546, BD), SMURF1 1:1000 (45-K, Santa Cruz), Myc 1:5000 (9E10, Santa Cruz), HA 1:5000 (Y11, Santa Cruz), GFP 1:5000 (FL, Santa Cruz) and GAPDH 1:10,000 (MAB374, Millipore).

Techniques: Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, Derivative Assay, Transfection, Expressing, Construct, Immunoprecipitation, Immunofluorescence, Clinical Proteomics, Membrane, Software, Binding Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Real-time PCR result from RT4 cells showing mRNA expression levels of genes indicated; error bars represent ± SD, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. B Immunoblot result showing EMT markers protein expression changes in RT4 cells upon TRAF4 knockdown. C RT4 cell colonies imaged by brightfield (top panels) or after exposure to CellMask™ Orange plasma membrane stain (bottom panels), scale: 25μM. D Images depicting RT4 spheroids made from control (empty vector pLKO) and TRAF4 knockdown cells (sh5), scale: 200μM. The graph represents circularities calculated from five independent spheroids of different sizes. Error bars represent ± SD, ** P ≤ 0.01 calculated using two-tailed student’s t test. E Real-time PCR result from HT1376 cells showing mRNA expression of the genes indicated; error bars represent ± SD, * P ≤ 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. F Immunoblot result showing EMT markers protein expression changes in HT1376 cells upon TRAF4 knockdown. G Representative images of transwell assays performed on HT1376 cells, cells were stained with crystal violet, scale: 200μM. H Quantification of number of migrated cells from four random fields; error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test.

Article Snippet: The following antibodies were used: TRAF4 1:2000 (D1N3A, CST), E-cadherin 1:1000 (Cat no. 610181, BD), N-cadherin 1:1000 (Cat no. 610920, BD), Vimentin 1:5000 (CST), SLUG 1:1000 (C19G7, CST), SNAIL 1:1000 (C15D3, CST), Flag 1:5000 (M2, Sigma Aldrich), phospho-Serine 1:1000 (612546, BD), SMURF1 1:1000 (45-K, Santa Cruz), Myc 1:5000 (9E10, Santa Cruz), HA 1:5000 (Y11, Santa Cruz), GFP 1:5000 (FL, Santa Cruz) and GAPDH 1:10,000 (MAB374, Millipore).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test, Western Blot, Knockdown, Clinical Proteomics, Membrane, Staining, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Immunoblot result of RT4 control (empty vector pLKO) and TRAF4 knockdown cells (sh4 and sh5) probed with the indicated antibodies. B Immunoblot result of HT1376 control (empty vector pLKO) and TRAF4 knockdown cells (sh4) probed with the indicated antibodies. Numbers represent relative quantification of SMURF1 levels with respect to GADPH. C Real-time PCR result showing SMURF1 mRNA expression levels in RT4 and HT1376 (control and TRAF4 knockdown) cells; error bars represent ± SD, n.s. indicates non-significant P value. D Immunoblot result from 293T cells transfected with the indicated plasmids. E Ubiquitination assay performed in 293T cells with Flag antibodies, over-expressing the indicated plasmids. Cells were treated with MG132 (2μM) overnight prior to lysis. Representative result from three independent experiments. F Real-time PCR showing Smurf1 mRNA levels in MBT-2 control (pLKO vector) and Smurf1 knockdown cells (sh1 and sh2); error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test. G Representative images of transwell assay performed on MBT-2 control and Smurf1 knockdown cells, which were stained with crystal violet, scale: 200μM. H Quantification of number of migrated cells from four random fields, error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test. I Graph showing relative wound density from Incucyte, images were obtained every 1 hour after wound was produced, T24 cells were treated with either DMSO or SMURF1 inhibitor, A01 (10μM); error bars represent ± SEM, *** P ≤ 0.001 calculated using two-tailed student’s t test. Representative result from three independent experiments . J Representative images from graph in I, brown area represents the cell coverage and grey area is the wound produced and remaining. K MTS assay performed on T24, cells were treated with either control (DMSO) or SMURF1i, A01 at 5μM. Absorbance was read at the indicated time points; error bars represent ± SD from three sample replicates, * P ≤ 0.05 calculated using two-tailed student’s t test.

Article Snippet: The following antibodies were used: TRAF4 1:2000 (D1N3A, CST), E-cadherin 1:1000 (Cat no. 610181, BD), N-cadherin 1:1000 (Cat no. 610920, BD), Vimentin 1:5000 (CST), SLUG 1:1000 (C19G7, CST), SNAIL 1:1000 (C15D3, CST), Flag 1:5000 (M2, Sigma Aldrich), phospho-Serine 1:1000 (612546, BD), SMURF1 1:1000 (45-K, Santa Cruz), Myc 1:5000 (9E10, Santa Cruz), HA 1:5000 (Y11, Santa Cruz), GFP 1:5000 (FL, Santa Cruz) and GAPDH 1:10,000 (MAB374, Millipore).

Techniques: Western Blot, Control, Plasmid Preparation, Knockdown, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing, Transfection, Ubiquitin Proteomics, Lysis, Two Tailed Test, Transwell Assay, Staining, Produced, MTS Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Immunoblot showing T24 cells stably expressing either control vector (myc-tag), TRAF4 or catalytically inactive TRAF4 mutant (C/A: cysteine to alanine at residue C18). B Representative images of transwell assays performed on T24 cells, cells were stained with crystal violet, scale: 100μM. C Quantification of number of migrated cells from four random fields, error bars represent ± SD, ** P ≤ 0.01, *** P ≤ 0.001 calculated using two-tailed student’s t test. D Graph showing relative wound density result from Incucyte. Representative result from three independent experiments; error bars represent ± SEM, *** P ≤ 0.001 calculated using two-tailed student’s t test. E Representative images from graph in D, brown area represents the cell coverage and grey area is the wound produced and remaining. F MTS assay performed with either control or TRAF4 stably expressing T24 cells. Absorbance was read at the indicated time points; error bars represent ± SD from three sample replicates, *** P ≤ 0.001 calculated using two-tailed student’s t test. G Immunoblot result showing MBT-2 cells stably expressing either control (empty vector with Myc-tag) or Myc-TRAF4. H Representative images of transwell assays performed on MBT-2 control and TRAF4 over-expressing cells, which were stained with crystal violet, scale: 200μM. I Quantification of number of migrated cells from four random fields, error bars represent ± SD, *** P ≤ 0.001 calculated using two-tailed student’s t test.

Article Snippet: The following antibodies were used: TRAF4 1:2000 (D1N3A, CST), E-cadherin 1:1000 (Cat no. 610181, BD), N-cadherin 1:1000 (Cat no. 610920, BD), Vimentin 1:5000 (CST), SLUG 1:1000 (C19G7, CST), SNAIL 1:1000 (C15D3, CST), Flag 1:5000 (M2, Sigma Aldrich), phospho-Serine 1:1000 (612546, BD), SMURF1 1:1000 (45-K, Santa Cruz), Myc 1:5000 (9E10, Santa Cruz), HA 1:5000 (Y11, Santa Cruz), GFP 1:5000 (FL, Santa Cruz) and GAPDH 1:10,000 (MAB374, Millipore).

Techniques: Western Blot, Stable Transfection, Expressing, Control, Plasmid Preparation, Mutagenesis, Residue, Staining, Two Tailed Test, Produced, MTS Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Graph showing differences in enrichment scores when TRAF4 is over-expressed in T24 cells. Gene signatures of eleven major cancer associated signalling pathways were considered for analysis. B Venn diagram showing number of genes which are up-regulated in two independent hairpins targeting TRAF4 in HT1376 (pink) and down-regulated with stable over-expression of TRAF4 in T24 (green) compared to their respective controls. 252 genes in the middle represents the reciprocally affected common genes. Results were obtained from four independent replicates for each sample. C Venn diagram showing number of genes which are down-regulated in two independent hairpins targeting TRAF4 in HT1376 (green) and up-regulated with stable over-expression of TRAF4 in T24 (pink) compared to their respective controls. 96 genes in the middle represents the reciprocally affected common genes. Results were obtained from four independent replicates for each sample. D Heatmap showing the common mis-regulated genes within the BMP, NF-κB and EMT gene signatures in both cell lines. E Real-time PCR result showing mRNA expression levels of the indicated genes in control (empty vector myc-tag) vs TRAF4 over-expressing T24 cells upon stimulation of BMP6 (50ng/ml) for 1 hour, error bars represent ± SD, * P ≤ 0.05, ** P ≤ 0.01, calculated using two-tailed student’s t test. F Real-time PCR showing mRNA expression levels of ID1, ID2 and ID3 in the indicated cell lines. G Graph showing relative wound density result from Incucyte, images were obtained every 1 hour after wound was produced, T24 cells were treated with BMP6 (50ng/ml); error bars represent ± SEM, *** P ≤ 0.001 calculated using two-tailed student’s t test. H Representative images from graph in G, brown area represents the cell coverage and grey area is the wound produced and remaining. I MTS assay performed with T24 cells, either control and stimulated with BMP6 (50ng/ml). Absorbance was read at the indicated time points; error bars represent ± SD from three sample replicates, ** P ≤ 0.01 calculated using two-tailed student’s t test.

Article Snippet: The following antibodies were used: TRAF4 1:2000 (D1N3A, CST), E-cadherin 1:1000 (Cat no. 610181, BD), N-cadherin 1:1000 (Cat no. 610920, BD), Vimentin 1:5000 (CST), SLUG 1:1000 (C19G7, CST), SNAIL 1:1000 (C15D3, CST), Flag 1:5000 (M2, Sigma Aldrich), phospho-Serine 1:1000 (612546, BD), SMURF1 1:1000 (45-K, Santa Cruz), Myc 1:5000 (9E10, Santa Cruz), HA 1:5000 (Y11, Santa Cruz), GFP 1:5000 (FL, Santa Cruz) and GAPDH 1:10,000 (MAB374, Millipore).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Control, Plasmid Preparation, Two Tailed Test, Produced, MTS Assay

Journal: bioRxiv

Article Title: TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

doi: 10.1101/2020.10.12.335588

Figure Lengend Snippet: A Luciferase reporter assay in 293T transfected with BRE-luciferase reporter, SV40 Renilla and either empty vector control or TRAF4, transfected cells were stimulated overnight with BMP6 (50ng/ml) and/or TNF-α (10ng/ml) where indicated, error bars represent ± SD, * P ≤ 0.05, *** P ≤ 0.001, calculated using two-tailed student’s t test, n.s. indicates non-significant P value. Representative result from three independent experiments. B Real-time PCR result showing mRNA expression levels of the indicated genes in control (empty vector myc-tag) vs TRAF4 over-expressing T24 cells upon stimulation of BMP6 (50ng/ml) and/or TNF-α (10ng/ml) where indicated for 1 hour. Error bars represent ± SD, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, calculated using two-tailed student’s t test. C Regression analysis showing correlation between TRAF4 expression levels (scores) and pSMAD1/5/8 scores from bladder cancer patients. Pearson’s test was used to determine the correlation between TRAF4 and pSMAD1/5/8 scores. D Regression analysis showing correlation between TRAF4 expression levels (scores) and p-p65 scores from bladder cancer patients. Pearson’s test was used to determine the correlation between TRAF4 and p-p65 scores. E Representative images of continuous sections of tissue microarray samples probed with the indicated antibodies using fluorescent immunohistochemistry. The magnified insets for pSMAD1/5/8 shows nuclear staining. F Schematic representation of TRAF4 signalling dynamics between epithelial and mesenchymal bladder cancer cells. Ub denotes Ubiquitin, Me is methylated promoter DNA and P stands for Phosphorylation of S334 site.

Article Snippet: The following antibodies were used: TRAF4 1:2000 (D1N3A, CST), E-cadherin 1:1000 (Cat no. 610181, BD), N-cadherin 1:1000 (Cat no. 610920, BD), Vimentin 1:5000 (CST), SLUG 1:1000 (C19G7, CST), SNAIL 1:1000 (C15D3, CST), Flag 1:5000 (M2, Sigma Aldrich), phospho-Serine 1:1000 (612546, BD), SMURF1 1:1000 (45-K, Santa Cruz), Myc 1:5000 (9E10, Santa Cruz), HA 1:5000 (Y11, Santa Cruz), GFP 1:5000 (FL, Santa Cruz) and GAPDH 1:10,000 (MAB374, Millipore).

Techniques: Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Control, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Microarray, Immunohistochemistry, Staining, Ubiquitin Proteomics, Methylation, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Primer sequences for the genes to validate the microarray analysis by RT-PCR.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Microarray

Journal: International Journal of Molecular Medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Effect of XBJ on the mRNA expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue. Groups of mice were challenged with CLP and treated with XBJ 24 h later. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Representative RT-PCR shows the level of Tollip, IRAK1, TLR4, NF-κB65, and TRAF6 expression in the four rat groups. M, marker; A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Control

Journal: International Journal of Molecular Medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Administration of XBJ enhanced the expression of Tollip protein protein, and inhibition TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Statistical summary of the densitometric analysis of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ** P<0.01 vs. the normal control group and sham operation group. # P<0.05, ## P<0.01 vs. the control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Immortal activated human hepatic stellate cells generated by ectopic telomerase expression.

doi: 10.1038/labinvest.3780426

Figure Lengend Snippet: Figure 2. Introduction of human telomerase reverse transcriptase (hTERT) in human HSCs leads to telomerase activity and permits maintenance of telomere length. Human HSCs were infected with a retrovirus encoding hTERT or a retroviral vector expressing only the neomycin resistance gene after 9 days in culture. Cells were selected in G418. A, The expression of hTERT was analyzed by semiquantitative RT-PCR using 1 g of total RNA. cDNA was analyzed from vector control–infected HSCs (vector) and from hTERT-infected HSCs (poly- clonal and single-cell clone 14). Telomerase-positive Hela cells and telomerase-negative primary human dermal fibroblasts (HDF) served as positive and negative control, respectively. Ubiquitin (below) was amplified to confirm equal amount of mRNA was present in each sample. B, Telomerase activity was measured in HSCs infected with a control vector (vector) or a vector expressing hTERT (polyclonal HSCs and HSCs from clone 14) by the telomeric repeat amplification protocol (TRAP) assay. For each sample 105

Article Snippet: RNA Isolation and RNase Protection Assay RNA was isolated from subconfluent HSCs using an RNA extraction kit (Qiagen) according to the manufacturer’s protocol.

Techniques: Reverse Transcription, Activity Assay, Infection, Retroviral, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Negative Control, Ubiquitin Proteomics, Amplification, TRAP Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Immortal activated human hepatic stellate cells generated by ectopic telomerase expression.

doi: 10.1038/labinvest.3780426

Figure Lengend Snippet: Figure 5. Gene expression patterns in activated human HSCs and telomerase-positive HSCs. A, Microarray analysis of gene expression in activated human HSCs and telomerase-positive HSCs was performed. The values of all genes in activated human HSCs (x axis) and telomerase-positive HSCs (y axis) were compared using scatter plot analysis. The upper and lower diagonals represent 2-fold higher gene expression in telomerase-positive and activated human HSCs, respectively. B, Uninfected and vector control–infected HSCs, telomerase- positive polyclonal HSCs, and HSCs from clone 14 were cultured on plastic. Total RNA (5 g) was subjected to RNase protection assay with riboprobes specific for human collagen 1(I) and human GAPDH mRNA. Migration of the protected bands is indicated. C, Western blot analysis was performed using whole cell extracts obtained from uninfected HSCs, vector control–infected HSCs, telomerase-positive polyclonal HSCs, and HSCs from clone 14. Proteins (10 g) were separated in a 12% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for SMA using monoclonal anti-smooth muscle -actin antibody.

Article Snippet: RNA Isolation and RNase Protection Assay RNA was isolated from subconfluent HSCs using an RNA extraction kit (Qiagen) according to the manufacturer’s protocol.

Techniques: Gene Expression, Microarray, Plasmid Preparation, Control, Infection, Cell Culture, Rnase Protection Assay, Migration, Western Blot, SDS Page, Membrane

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Immortal activated human hepatic stellate cells generated by ectopic telomerase expression.

doi: 10.1038/labinvest.3780426

Figure Lengend Snippet: Figure 6. Culturing telomerase-positive HSCs on a basement membrane-like matrix reverts them to a quiescent phenotype and down-regulates their collagen 1(I) mRNA. Telomerase-positive HSCs from clone 14 were plated in matrigel, a basement membrane-like matrix, at a density of 4 to 6105 per 60-mm dish. A, Phase microscopy demonstrates cluster formation without visible spreading of telomerase-positive HSCs in matrigel (original magnification, 100). B, HSCs were maintained in matrigel in hormonally defined medium (HDM) containing 2% FCS for 15 days. As controls telomerase-positive HSCs from clone 14 were cultured on plastic for 15 days after plating, either in HDM containing 2% FCS or in regular growth medium containing 10% FCS. RNase protection assay with collagen 1(I) and GAPDH gene-specific riboprobes and RNA (3.5 g) extracted from HSCs was performed. Migration of the protected bands is indicated. Expression of collagen 1(I) mRNA was normalized to GAPDH mRNA, and the ratios are indicated. The results are expressed relative to HSCs cultured on plastic in HDM containing 2% FCS.

Article Snippet: RNA Isolation and RNase Protection Assay RNA was isolated from subconfluent HSCs using an RNA extraction kit (Qiagen) according to the manufacturer’s protocol.

Techniques: Membrane, Microscopy, Cell Culture, Rnase Protection Assay, Migration, Expressing