Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 7. Blocking Src activity reduces proteinuria in msuPAR2-Tg mice. (A) Glomerular Src activity was determined by immunofluorescent staining of the kidney cryosections with p-Src monoclonal antibody (green). Podocin was used as a podocyte marker (red). Shown are the representative glomeruli with p-Src immunofluorescent staining. As indicated by the p-Src staining intensity, glomerular Src phosphorylation was readily observed in the kidney sections from msuPAR2-Tg, but not from other investigated mice. Scale bars: 20 μm. (B) Treatment with Src inhibitors lowered proteinuria in msuPAR2-Tg mice. Both PP1 and Src inhibitor 1 (5 mg/kg) were administered into the randomly grouped msuPAR2-Tg mice intraperitoneally, with vehicle controls receiving the same amount of DMSO. n = 8 Src inhibitor group; n = 7 vehicle control group. At 72 hours after treatment, proteinuria was significantly reduced by Src inhibitor treatment. Two-way ANOVA; data were log-transformed to normal distribution. **P < 0.01.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Blocking Assay, Activity Assay, Staining, Marker, Phospho-proteomics, Control, Transformation Assay

Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 8. Glomerular c-Src activity is increased in human FSGS kidney. Immunofluorescent staining with p–c-Src antibody was performed for the frozen sections of deidentified human kidney biopsies. Synaptopodin was used as a podocyte marker. Shown is a representative of 4 batches of immu- nostaining. While a minimal amount of c-Src phosphorylation was observed in the glomeruli of healthy donors (n = 3), glomerular p–c-Src intensity was increased in 6 out of 10 FSGS patients. Overlap of p–c-Src (green) and synpo (red ) indicates that p–c-Src was local- ized in podocytes. Of note, only 11 out of 24 observed glomeruli were positive for p–c-Src, from which 64% were focal, 36% globally but not evenly. In contrast, the increase of pSrc was not observed in other glomerular diseases, including SLE (n = 2), MPGN (n = 2), and MCD (n = 4). Scale bar: 20 μm. NT, normal kidney tissue.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Activity Assay, Staining, Marker, Phospho-proteomics

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 expression and prognosis in intrinsic subtypes of breast cancer . Box plots of JMJD6 expression demonstrate (A) higher JMJD6 probeset intensity (log2) in ER - tumors compared with ER + tumors, with P < 0.001 by Mann-Whitney Rank Sum test; and (B) highest JMJD6 probeset intensity (log2) in claudin-low and basal subtypes, followed by HER2 -enriched and LumB subtypes, and lowest in LumA subtypes. The Dunn method was used to perform pairwise multiple comparison among the subtypes. * P < 0.05 between a pair of subtypes. Dots of boxplots (A, B) represent outliers in the 90 th and 10 th percentiles. Kaplan-Meier survival curves based on below-median (low) and above-median (high) JMJD6 expression are shown for (C) ER + patients, (D) patients of LumA subtype, (E) patients of LumB subtype, and (F) ER + , tamoxifen-treated patients. The numbers in the parentheses equal patient numbers in each group, and the log rank P value is indicated at the bottom left of each figure.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, MANN-WHITNEY, Comparison

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Hazard ratios of JMJD6 expression groups in various subtypes of breast cancer

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Nuclear JMJD6 protein expression is associated with high-grade breast tumors . Representative breast cancer cores from tissue microarray that were stained with JMJD6 antibody are shown. Low-grade (left panel), intermediate-grade (middle panel), and high-grade breast tumors (right panel) are shown (magnification, ×60). The table in the figure shows univariate analysis of JMJD6 and clinical parameters.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, Microarray, Staining

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Expression of JMJD6 enhances proliferation . (A) WST-1 assays using MCF-7-J1-OE clones (J1-C2, J1-C3, and J1-C7) showed increased proliferation over vector control cells (Vec). SiRNA-mediated knockdown of JMJD6 and resultant decrease in proliferation is shown in (B) MCF-7 and MDA-MB231. Four individual siRNAs (A, B, C, and D) were used: Sc, scrambled siRNA; and NT, non-transfected cells served as control. Inset in each panel shows protein levels of JMJD6 after siRNA transfection. β-actin immunoblots show equal protein loading in all lanes.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, Clone Assay, Plasmid Preparation, Control, Knockdown, Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 promotes motility in breast cancer cell lines . (A) Scatter phenotype was scored based on three levels of scattering (compact, loose, scatter) (upper panel). Bar graphs represent quantification of scattering of colonies by using the Student t test (lower panel). MCF-7 J1 OE clones had a higher percentage of scattered or loosely scattered clones than did the control Vec cells. (B) Wound-healing assay showed that MCF-7 J1 clones displayed higher motility in comparison to Vec (upper panel); quantification of wound closure as a ratio to the Vec is shown in the lower panel. * P ≤ 0.05 by Student t test in comparison to Vec control. (C) Boyden-chamber assay results of MDA-MB231 cells with JMJD6 siRNA-mediated knockdown were normalized to fold change in proliferation at Day 2 of WST-1 measurement. Two of three siRNA knockdowns of JMJD6 showed significantly decreased motility, and all siRNA knockdowns of JMJD6 showed decreased invasiveness. * P ≤ 0.05; ** P ≤ 0.001 by Student's t- test when compared with Sc siRNA.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Clone Assay, Control, Wound Healing Assay, Comparison, Boyden Chamber Assay, Knockdown

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: IPA analysis of JMJD6-regulated genes showed enrichment in cell-cycle function . (A) The heatmap (top panel) shows that hierarchic clustering of the genes changed after siRNA knockdown and overexpression of JMJD6 . The sample labels are on the top of the heat map. Two clusters representing JMJD6-induced and JMJD6-repressed genes were selected (middle heat-map panel) and subjected to IPA analysis. (B) Table shows a list of the top 10 functions that are enriched in cells with altered levels of JMJD6.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Knockdown, Over Expression

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 represses TGF-β2 expression . RT-qPCR assays of TGF-βs after JMJD6 siRNA-mediated knockdown are shown in (A) MCF-7 and (B) MDA-MB231. Consistently, T GF-β2 and TGF-β1 mRNA levels were upregulated on JMJD6 knockdown. (C) Immunoblots showed that the amount of secreted TGF-β2 protein in conditioned media was higher after JMJD6 siRNA-mediated knockdown in MCF-7 and MDA-MB231. (D) RT-qPCR assay showed that MCF7 J1 OE clones had a lower level of TGF-β1 and TGF-β2 transcripts than did the Vec cells. (E) Western blot showed a dramatic decrease in secreted TGF-β2 protein, in the MCF-7 J1 clones, as compared with the Vec cells. NS, a Ponceau-stained blot of the conditioned media to demonstrate protein loading. Student's t - test was performed by using scrambled siRNA versus JMJD6 siRNA and Vec cells versus MCF-7J1 clones. * P ≤ 0.05; ** P ≤ 0.005.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Clone Assay, Staining

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 expression affects SMAD phosphorylation . Immunoblots show that the levels of total SMAD2/3, phosphorylated SMAD2, and phosphorylated SMAD3 are decreased in MCF-7 J1-OE clones and increased in JMJD6 siRNA-mediated knockdown in MCF-7 and MDA-MB231. The numbers next to the SMAD2P/3P denote the expected sites of phosphorylation detected by the antibodies. NS, nonspecific bands from the blots, which indicate equal loading of the protein lysates.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, Phospho-proteomics, Western Blot, Clone Assay, Knockdown

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: TGF-β2 inhibits proliferation in MCF-7 . (A) Immunoblots showed that treatment of MCF-7 by recombinant human (rh) TGF-β2 (5 ng/ml) resulted in enhanced Smad2 phosphorylation, which could be effectively nullified by SB431542. (B) Treatment of JMJD6 siRNA-transfected MCF-7 cells with SB431542 (10 μ M ) resulted in rescue of the proliferation defect. Ratio of OD at Day 3 to Day 1 in JMJD6 knockdown cells was normalized to the scrambled siRNA control. * P ≤ 0.05; ** P ≤ 0.005 between DMSO and SB431542 treatments. (C) Treatment of MCF-7 J1-OE and Vec cells with rhTGF-β2 resulted in decreased proliferation, as measured by WST-1 assay. ** P ≤ 0.001 between BSA and rhTGF-β2 treatments.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Western Blot, Recombinant, Phospho-proteomics, Transfection, Knockdown, Control, WST-1 Assay

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Decrease of cyclin E2 protein level in JMJD6 knockdown . Western blot detection of G 1 cell-cycle cyclins is shown. A decrease in cyclin E2 (CCNE2) level with decrease in JMJD6 levels was evident in MCF-7 and MDA-MB231. Cyclin E1 (CCNE1) and cyclin D1 (CCND1) displayed an inconsistent change on JMJD6 siRNA transfection in both MCF-7 and MDA-MB231 cells.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Knockdown, Western Blot, Transfection

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Clinical association of JMJD6 with TGF-β2 . Pearson correlation of clinical expression of JMJD6 with TGF-β2 suggests a negative correlation with TGF-β2 ( P = 9.13 × 10 -8 ). Scatterplot shows the normalized JMJD6 expression of each patient sample with the corresponding normalized TGF-β2 expression in the clinical dataset of 2,034 breast cancer patients.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing