Journal: Medicina

Article Title: Meta-Analysis of Survival Effects of Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1)

doi: 10.3390/medicina58121867

Figure Lengend Snippet: Characteristics included for the study of ROR1.

Article Snippet: H. Chang (2015) [ ] , Republic of Korea , Gastric cancer , 424 , IHC , Rabbit polyclonal antibody (1:25; Abcam) , Staining > 50% , - , 0.8 (0.53–1.21) p = 0.189.

Techniques: Staining, Expressing, Fluorescence

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and nuclear localization in cancer cell spheres. a shScr and shFGFR4 MDA-MB-453 cell spheres were cultured under non-adherent conditions (10% or 2% FBS), and subjected to immunoblotting. Arrowhead; cleaved N-terminal MST1/2 (in 2% FBS), brackets highlight the fragments of autoactivated MST1/2. b MDA-MB-453 cell spheres were treated with 100 n m BLU9931 for 15 min, and subjected to immunoblotting. c , d shScr and shFGFR4 MDA-MB-453 and ZR-75.1 spheres were analyzed for MST1 expression by c immunofluorescence, and d MST1 nuclear/cytoplasmic ratio was quantified ( n = 4–6 MDA-MB-453 spheres, ≥ 6 microscopic fields/sphere; n = 2–3 ZR-75.1 spheres, ≥ 8 microscopic fields/ sphere; mean ± SEM of two independent experiments. Scale bar 10 µm. e shScr and shFGFR4 MDA-MB-453 cells were transfected with indicated siRNAs before sphere formation, cultured under non-adherent conditions (1% FBS) for 48 h, and subjected to immunoblotting

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Immunofluorescence, Transfection

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: MST1-Y433F phosphosite mutant restores MST1/2 activation in FGFR4 expressing cancer cells. a MDA-MB-231 cells co-transfected with FGFR4 (R) and wild-type or phosphosite mutant MST1-Y433F were subjected to immunoblotting as indicated. Ratio of pMOB1/MOB1 is indicated below the immunoblot panel. b T47D cells (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid for 1 h before cell lysis, and subjected to immunoblotting. See corresponding T47D immunoblots without okadaic acid in Fig. S4C. c T47D cells with indicated siRNAs, and (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid as above, and subjected to immunoblotting. a–c Brackets and arrowhead indicate the activated pMST1/2 fragments. N = 2 independent repeats

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Phospho-proteomics, Mutagenesis, Activation Assay, Expressing, Transfection, Western Blot, Lysis

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 substrate screen identifies tyrosine-phosphorylated Hippo pathway proteins including MST1/2. a Scheme of the substrate screen with recombinant FGFR4 kinase domain. b Top 10 FGFR4 substrates ranked by the Z-score include Hippo pathway -associated proteins (yellow). See Table S1 for the full substrate list. c , d MST1/2 are tyrosine phosphorylated by FGFR4 in COS-1 cells. Flag-tagged MST1/2 were immunoprecipitated after transfection of MST1 and MST2 alone or in combination with FGFR4 G388 (G), or R388 (R) kinase (wt), or kinase-dead (KD) variants, and detected by immunoblotting. e MST1 immunoprecipitates from COS-1 cells co-transfected with FGFR4 (R)-wt or FGFR4 (R)-KD (See Fig. S1A) were trypsin digested and subjected to phoshopeptide enrichment prior to LC-MS/MS analysis ( N = 3) that identified phosphorylated Y433 (red) on MST1 only with FGFR4 (R)-wt, and phosphorylated S410 (green) only with FGFR4 (R)-KD

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Recombinant, Immunoprecipitation, Transfection, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: List of MST1 phoshopeptides identified by mass spectrometry

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Sequencing

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 is overexpressed in HER2 + , MST1/2 low breast cancer cells. a , b FGFR4 and HER2 expression in luminal MDA-MB-453, ZR-75.1, and BT474, MCF7, and T47D, and five triple-negative breast cancer cell lines by a immunoblotting and b immunofluorescence. Scale bar 20 μm. c MST1, MST2, and YAP/TAZ expression in these cell lines, detected by immunoblotting ( N = 3)

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and cleavage in HER2 + breast cancer cells. a , b MDA-MB-453 cells transfected with indicated siRNAs were subjected to immunoblotting for a T183/180 phosphorylated MST1/2, and b MST1 and MST2. Note cleaved ~ 37 kDa MST1/N in FGFR4 knockdown cells (arrowhead). Thin gray line indicates cropping to leave out irrelevant sample lane; see uncropped immunoblots in Fig. S8. c MDA-MB-453 cells transduced with indicated shRNAs were transfected with siScr or siFGFR4 siRNA to 3’UTR before transfection of mock or FGFR4 (R) or (G) overexpression plasmid for a rescue experiment. Lysates were subjected to immunoblotting as indicated. Brackets indicate the cleaved MST1 and MST2 fragments. See Fig. S2A for phopsho-FRS2α and short exposure of MST1. d MDA-MB-453 and ZR-75.1 cells were transduced with indicated si/shRNAs; upper, indicated immunoblots of lysates; lower, quantification of pMOB1/MOB1 ratio, N = 3, mean ± SEM; * P < 0.05. For MST1/2 knockdown e ZR-75.1 and f MDA-MB-453 were transduced with shRNAs followed by transfection with siRNAs as indicated, and g BT474 cells were transfected with indicated siRNAs, and subjected to immunoblotting for pT183/180 MST1/2, MST1, MST2, and pMOB1 as indicated (in e arrowhead points to a full-length, bracket to the cleaved MST2) a–g . N = 3 independent repeats for all; except N = 2 in f and g

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Transfection, Western Blot, Knockdown, Transduction, Over Expression, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 counteracts MST1/2-mediated apoptosis. MDA-MB-453 cells transduced with shScr or shFGFR4 shRNAs were transfected with siRNA pools specific for FGFR4, MST1 or MST2, and analyzed for annexin V and propidium iodide (PI) binding by flow cytometry using two different gating strategies for data visualization. a Gating to populations P1 (smaller) and P2 (larger), and annexin V binding (FL1-A) histograms as a marker for early apoptotic cells. b Quantification (% of total, 100,000 events) of apoptosis based on double-positive (annexin V + PI) cells, including both early and late apoptotic stages. See Fig. S3B for representative contour plots and quadrant gating. Mean ± SD of triplicates shown, ** P < 0.01; (repeated three times; N = 3). FSC-A; forward scatter, and SSC-A; side scatter

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Transduction, Transfection, Binding Assay, Flow Cytometry, Marker

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 confers resistance to apoptotic modulators in comprehensive drug screen. a (Phospho)protein changes in TCGA RPPA data associated with FGFR4 upregulation in breast cancer, visualized using cBioPortal (RPPA score change in breast cancer tumors with and without alterations in FGFR4; (mean FGFR4 altered – mean FGFR4 unaltered) [ , ]. The most significantly up- and downregulated proteins are highlighted (pink dots); ERBB2, alternative name of HER2; PR, progesterone receptor. b–g Fibrin embedded single-cell suspensions of b–d MDA-MB-453 and e–g ZR-75.1 cells were treated with 100 n m BLU9931 and/or 30 ng/ml FGF1 over a 13–14-day culture, fixed, embedded into paraffin for sectioning, and subjected to immunohistochemistry for Ki67 and BAX expression. Positively stained vs. total number of cells per colony were counted ( N = 30, mean ± SD, ** P < 0.01). Scale bar 50 µm in b and e . b For comprehensive drug sensitivity testing ( N = 1), MDA-MB-453 cells were treated with 527 compounds in five-point dose either alone or in combination with specific FGFR4 inhibitor BLU9931. Dotplot showing the difference in DSS (drug sensitivity score) for cells in treatment combination with BLU9931 (100 n m ) versus single agent treatments. Negative values are compounds inducing larger decreases in viability as single agents; positive scores indicate compounds yielding larger decreases in viability in the presence of BLU9931. Colors demarcate compounds with similar class

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Predominant control of PDGF/PDGF receptor signaling in the migration and proliferation of human adipose‑derived stem cells under culture conditions with a combination of growth factors

doi: 10.3892/etm.2024.12444

Figure Lengend Snippet: Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

Article Snippet: After blocking with Blocking One-P reagent (cat. no. 05999-84; Nacalai Tesque, Inc.) for 60 min at room temperature, the membranes were incubated overnight at 4˚C with the following primary antibodies: Anti-phospho-Erk1/2 (1:1,000; cat. no. #4370; Cell Signaling Technology, Inc.), anti-Erk1/2 (1:1,000; cat. no. #4695; Cell Signaling Technology, Inc.), anti-phospho-PDGFRb (1:1,000; cat. no. GTX133525; GeneTex, Inc.), anti-PDGFRb (1:1,000; cat. no. 134491AP; Proteintech Group, Inc.), anti-phospho-c-Met (1:1,000; cat. no. 600401989S; Rockland Immunochemicals Inc.), anti-c-Met (1:1,000; cat. no. GTX631992; GeneTex, Inc.), anti-phospho-VEGFR2 (1:1,000; cat. no. CSBPA000703; Cusabio Technology, LLC), anti-VEGFR2 (1:1,000; cat. no. CSBPA008334; Cusabio Technology, LLC), anti-phospho-p38 MAPK (1:1,000; cat. no. #4511; Cell Signaling Technology, Inc.), anti-p38 MAPK (1:1,000; cat. no. #8690; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,000; cat. no. #4970; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Cell Culture, Incubation, SDS Page, Western Blot, Derivative Assay

Journal: EMBO Reports

Article Title: Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase

doi: 10.1038/s44319-025-00397-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-eIF4E (Boster) was used at 1:10000.

Techniques: SYBR Green Assay, Imaging, Recombinant, Sequencing, Software, Microscopy, Ion-Mobility Spectrometry, Mass Spectrometry

Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: A – D Kinetics of TrkB-FL downregulation. Primary cortical cultures were treated with 100 μM NMDA and its co-agonist 10 μM glycine (hereafter referred to as ‘NMDA’). Immunofluorescence ( A ) and immunoblotting ( B ) used a C-terminal (C-ter) isoform-specific antibody (TrkB-FL Ct) recognizing both the full-length protein (FL) and the intracellular fragment (f32). A Shows TrkB-FL (green) and nuclei (blue, DAPI stain). Arrowheads indicate varicosities in neuronal projections. Scale bar: 20 μm. Insets show cell body details for untreated cells and cells treated with NMDA for 120 min. B Compares the decrease in TrkB-FL and formation of f32 with PSD-95 downregulation, detected using a C-terminal antibody (PSD-95 Ct). Calpain activation was confirmed by the accumulation of characteristic spectrin breakdown products (BDPs; 150 and 145 kDa). Neuron-specific enolase (NSE) served as a loading control for protein normalization. C , D Quantification of normalized TrkB-FL and PSD-95 levels, shown relative to levels in the absence of NMDA (control). Data are represented as means ± SD. Statistical analysis: one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (** P < 0.01, *** P < 0.001, **** P < 0.0001; 0-90 min, n = 5; 120 min, n = 3). E , F Effect of dynasore preincubation (80 μM, 30 min) on TrkB-FL levels after 2 h NMDA treatment. Data are means ± SD ( n = 4); statistical analysis as above (* P < 0.05). G Sequences of the cell-penetrating neuroprotective peptide (MTFL 457 ) and control peptide (MTMyc). Both contain Tat amino acids (aa) 47–57 (italic), followed by rat TrkB-FL aa 457-471 (light blue) or c-Myc aa 408-421 (dark blue), respectively. H , I Effect of peptide preincubation (25 μM, 30 min) on TrkB-FL shedding and processing during NMDA treatment. Culture media were analyzed using an antibody against the TrkB-FL extracellular domain (panTrkB), which recognizes the ectodomains (ECDs) of all isoforms (TrkB-ECD), to assess receptor shedding by metalloproteinase (MP) activation. Total lysates were analyzed with the TrkB-FL Ct antibody to evaluate TrkB-FL calpain processing via f32 production. Relative TrkB-ECD levels are shown as means ± SD (0-4 h, n = 7; 6 h, n = 4). Statistical analysis: two-way ANOVA followed by Bonferroni post hoc test (** P < 0.01, **** P < 0.0001, comparing each peptide + NMDA vs. peptide alone; # P < 0.05, #### P < 0.0001, comparing MTMyc vs. MTFL 457 at each time point). J , K Effect of NMDA on total and cell-surface pY816-TrkB-FL levels. Cultures were preincubated with peptides as above, then treated briefly with NMDA (1 h) to minimize receptor degradation. Cell-surface proteins were biotin-labeled and precipitated, then compared to corresponding total lysates. Data are represented as means ± SD ( n = 4). Statistical analysis: two-way ANOVA followed by Bonferroni post hoc test ( n.s . = n ot significant; ** P < 0.01, comparing NMDA vs. no NMDA; ## P < 0.01, comparing MTMyc + NMDA vs. MTFL 457 + NMDA).

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Immunofluorescence, Western Blot, Staining, Activation Assay, Control, Labeling

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: Primer sequences in this paper

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: BRD7 interacts with YB1. a Coomassie blue staining of co-immunoprecipitation using anti-IgG or anti-flag antibodies in BRD7 overexpression HEK293T cells. b Quantification of YB1 expression in TCGA BRCA database ( n = 823) of different clinical types (Luminal, her2 positive and triple negative types). c Km-plot analysis of YB1 expression and survival of breast cancer patients consist of 1976 patients in YB1 low expression group and 1975 patients in YB1 high expression group. d Co-immunoprecipitation (top) using anti-flag antibodies in flag-BRD7 overexpressed of HEK293T, MDA231 and MCF7 cells and western blotting analysis of flag and YB1. Co-immunoprecipitation (down) using anti-HA antibodies in HEK293T, MDA231 and MCF7 cells of flag-BRD7 and HA-YB1 overexpression and western blotting analysis of HA and flag. e IF using anti-flag or anti-YB1 antibodies in MDA231 cells of flag-BRD7 overexpression. f Schematic illustration of different extents of brd7 mutants. g Co-immunoprecipitation using anti-flag antibodies in HEK293T cells co-transfected with HA-BRD7 deletion mutants and flag-YB1 and western blotting analysis of HA and flag. h Schematic illustration of different extents of YB1 mutants. i Co-immunoprecipitation using anti-flag antibodies in HEK293T cells co-transfected with flag-YB1 deletion mutants and HA-BRD7. Western blotting analysis of flag and HA

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques: Staining, Immunoprecipitation, Over Expression, Expressing, Western Blot, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: BRD7 induces ubiquitination degradation of YB1 depended on YB1 Ser102 phosphorylation. a Western blotting analysis of BRD7 and YB1 in MDA231 and MCF7 cells with BRD7 overexpression. b qPCR analysis of YB1 in BRD7 overexpression of MDA231 and MCF7 cells. c Western blotting analysis of Flag-BRD7 and YB1 in BRD7 overexpressed MDA231 cells treated with or without MG132 (20 μM) for 4 h. d Co-immunoprecipitation using anti-YB1 antibodies in MDA231 cells co-transfected Ub with flag-BRD7 or control and treated with or without MG132(20 μM) for 4 h. Western blotting analysis of Ub and YB1. e Western blotting analysis of BRD7 and p-YB1ser 102 in MDA231 and MCF7 cells transfected with BRD7. f Co-immunoprecipitation using anti-flag antibodies in HEK293T cells co-transfected by BRD7 along with either YB1 wild-type or YB1 mutant plus HA-ubiquitin for 48 h, treated with MG132(20 μM) for 4 h. Western blotting analysis of Ub, flag, HA, p-YB1 and GAPDH

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Over Expression, Immunoprecipitation, Transfection, Control, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: BRD7 inhibits the process of EMT. a GSEA analysis of microarray data from BRD7 overexpressed (left), YB1 knockdown (middle) or YB1 overexpressed cells (right) and control. b qPCR analysis of E-cadherin, Claudin1, vimentin and Snail in MDA231 cells with BRD7 overexpression. Data represent means ± SEMs. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. c qPCR analysis of E-cadherin, Claudin1, vimentin and Snail in MDA231 cells with BRD7 inhibition. Data represent means ± SEMs. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. d Immunoblots of BRD7, E-cadherin, Claudin1, vimentin and Snail in MDA231 and MCF7 cells with BRD7 overexpression or in MDA231 cells with BRD7 knock-down

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques: Microarray, Knockdown, Control, Over Expression, Inhibition, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: YB1 antagonizes the inhibitory effect of BRD7 on cell proliferation, migration and invasion. a and b CCK8 analysis of cell proliferation in MDA231 and MCF7 cells stably with BRD7 overexpression, BRD7 and YB1 simultaneous overexpression or control group. Data represent means ± SDs. *, p < 0.01. c Scratch wound healing analysis of cell migration in MDA231 cells with BRD7 overexpression, BRD7 and YB1 simultaneous overexpression or control. Quantification of wound recovery rate of the three groups (right). Data represent means ± SEMs. **, p < 0.01; ***, p < 0.001. d Matrigel invasion analysis of cell invasive capabilities in MDA231 and MCF7 cells with BRD7 overexpression, BRD7 and YB1 simultaneous overexpression or control. Data represent means ± SDs. **, p < 0.01. e Western blotting analysis of the expression of BRD7, YB1, E-cadherin, Claudin1, vimentin, Snail and p21 in BRD7 overexpression and YB1 restoration cells

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques: Migration, Stable Transfection, Over Expression, Control, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: BRD7 suppresses tumor growth and reduces lung metastasis through YB1 in vivo. a , b and c Tumor volume, image and tumor weight of nude mice with MDA231 cells in xenograft model, n = 5 mice per group. Data represent means ± SDs. **, p < 0.01. d Representative image of macroscopic mouse lung tissue in the metastatic tumor model, n = 11 mice per group. e Representative image of lung metastasis samples by H&E staining is shown in control, BRD7 overexpression and YB1 restoration group. Red arrows indicate metastatic tumors, scale bar, 200 μm. The number of metastatic lung nodules of every mouse per group were counted in microscopy. Data represent means ± SDs. **, p < 0.01; ***, p < 0.001. f Primary tumor samples for IHC analysis of the expression of BRD7, YB1, Ki67 in control, BRD7 overexpression and YB1 restoration group, scale bar, 20 μm. h Primary tumor samples for IHC analysis of the expression of EMT markers E-cadherin and vimentin, scale bar, 20 μm

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques: In Vivo, Staining, Control, Over Expression, Microscopy, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: BRD7 is negatively correlated with YB1 in breast cancer. a YB1 expression was determined in normal ( n = 43) and tumor samples ( n = 220) by IHC. b YB1 expression in different T stages of breast cancer. c and d Kaplan-Meier curves showed the overall survival of breast cancer patients. High or low expression of YB1, and low BRD7 plus high YB1 level and high BRD7 plus low YB1 level. e The correlation between BRD7 and YB1 was performed based on chi-square test. f Schematic representation of molecular mechanism of BRD7 in suppressing tumor growth and metastasis

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: BRD7 suppresses invasion and metastasis in breast cancer by negatively regulating YB1-induced epithelial-mesenchymal transition

doi: 10.1186/s13046-019-1493-4

Figure Lengend Snippet: The association between BRD7, YB1 expression and clinicopathologic features of breast cancer

Article Snippet: Antibodies against anti-BRD7 (51009–2-AP, proteintech, 1:1000 dilution), anti-YB1 (CY5462, Abways Technology, 1:1000 dilution), anti-Phospho-YB1 (Ser102) Antibody (CSB-PA204680, Cusabio, 1:1000 dilution), anti-HA (561–7, MBL, 1:1000 dilution), anti-Flag (F3040, Sigma-Aldrich, 1:1000 dilution), anti-Vimentin (ARG66302, arigo, 1:1000 dilution, 1:200 dilution for IF), anti-Snail (C15D3, CST, 1:1000 dilution), anti-E-cadherin (24E10, CST, 1:1000 dilution), anti-Claudin1 (D5H1D, CST, 1:1000 dilution, 1:50 dilution for IF) and anti-GAPDH (10494–1-AP, proteintech, 1:20000 dilution).

Techniques: Expressing