Journal: Development (Cambridge, England)

Article Title: LRP2/megalin is required for patterning of the ventral telencephalon.

doi: 10.1242/dev.01580

Figure Lengend Snippet: Fig. 9. Megalin mediates binding and cellular catabolism of BMP4. (A) Surface plasmon resonance (SPR) analysis of binding of 0.5 µmol/l BMP4 but not of 0.5 µmol/l BMP5 or WNT1- conditioned medium to purified megalin immobilized on the sensor chip surface (see Materials and methods for details). As a positive control, binding of 1 µmol/l receptor-associated protein (RAP) to megalin was tested. (B) Megalin-expressing BN16 cells mediate uptake and lysosomal degradation of 125I-BMP4. Degradation of BMP4 can be blocked by chloroquine (200 µmol/l) or the receptor-antagonist RAP (100 µg/ml).

Article Snippet: BIAcore analysis and cell culture experiments Carrier-free preparations of recombinant human BMP4 and BMP5 were purchased from Research and Development (www.RnDSystems.com).

Techniques: Binding Assay, SPR Assay, Positive Control, Expressing

Journal: Development (Cambridge, England)

Article Title: Bone morphogenetic proteins specify the retinal pigment epithelium in the chick embryo.

doi: 10.1242/dev.02884

Figure Lengend Snippet: Fig. 2. Comparison of the Sox10, Mitf and Bmp expression patterns during the initial stages of chick eye development. (A) Schematic illustrating the location of the section in H, showing the nasal part of the optic vesicle at stage 10/11. (B) Bmp4 expression in the surface ectoderm overlying the optic vesicle following whole-mount in situ hybridisation (13 somites). The optic primordium is shown at stage 8 (6 somites; C,F,I,L,O), at stage 9 (7-9 somites; D,G,J,M,P) and stage 10 (10-12 somites; E,H,K,N,Q). (C) At stage 8, Sox10-positive neural crest cells are observed in the dorsal neural folds of the prosencephalon (arrows). (D) Sox10-expressing neural crest cells are detected in the dorsal-most region of the prosencephalon (arrows) and no transcripts are detected distally. (E) Sox10 expression is detected in neural crest cells overlying the dorsal part of the optic vesicle (arrows). (F) At stage 8, Mitf expression is not observed in the optic primordium (arrow). (G) Mitf expression is strongest in the distal part of the optic vesicle at stage 9 (arrows). The arrowheads indicate neural crest cells dorsally. (H) At stage 10, Mitf expression is observed in the presumptive RPE (dorsal optic vesicle) and in the distal part (arrows). See A for the location of this section; see also R,S,T. (I) Bmp7 expression is observed in the ectoderm overlying the optic primordium at stage 8 (arrows), and diffuse expression is detected in the neural folds (arrowheads). (J) Parallel section of the embryo shown in G. At stage 9, strong Bmp7 expression is observed in the overlying ectoderm (arrows). Bmp7 transcripts are also detected in the dorsal ectoderm that covers the neural crest cells (arrowheads). (K) Bmp7 expression in the ectoderm overlying the distal region of the optic vesicle at stage 10 (arrows). Transcripts are still observed in the ectoderm overlying the mesenchyme (arrowhead). (L) At stage 8, Bmp4 transcripts are detected in the overlying ectoderm (arrow) and in the neural folds (arrowheads). (M) Strong Bmp4 expression is detected in the ectoderm overlying the distal portion of the optic vesicle at stage 9 (arrows). Weak or no expression is observed in the dorsal-most ectoderm overlying the mesenchymal cells (arrowheads). (N) At stage 10, Bmp4 expression is still strong in the ectoderm overlying the distal portion of the optic vesicle (arrows), whereas weak expression is observed in the ectoderm overlying the surrounding mesenchyme (arrowhead). Note that Bmp4 expression appears to be stronger in the ectoderm overlying the dorsal portion of the optic vesicle. (O) At stage 8, Bmp5 expression is strong in the dorsal midline, the neural folds (arrowheads). Transcripts appear to be absent from the overlying ectoderm. (P) Bmp5 expression weakens in the dorsal midline at stage 9 (arrowhead). (Q) No Bmp5 transcripts are detected in the neuroepithelium of the chick optic vesicle and surrounding tissues (arrow) at stage 10. (R) Nasal region of the optic vesicle at stage 10. Mitf expression in the distal and dorsal part of the optic vesicle (arrowheads). (S) Higher magnification of the Mitf expression pattern in the more-temporal region of the optic vesicle shown in H. Mitf expression is detected in both the dorsal and distal region of the optic vesicle (arrowheads), although expression weakens ventrally (arrow). (T) In the most-temporal region of the optic vesicle, Mitf expression is downregulated in the disto-ventral region (arrow).

Article Snippet: A 2 l drop of recombinant mouse BMP5 or BMP4 (0.7 mg/ml or 1 mg/ml; R&D Systems) was placed in a Petri dish and about eight drops (10 l each) of distilled water were placed around it to keep it from evaporating.

Techniques: Comparison, Expressing, In Situ, Hybridization

Journal: Development (Cambridge, England)

Article Title: Bone morphogenetic proteins specify the retinal pigment epithelium in the chick embryo.

doi: 10.1242/dev.02884

Figure Lengend Snippet: Fig. 3. Effects of BMP and noggin application on the Wnt2b expression pattern during early stages of eye development. (A) Schematic of a stage 10/11 chick embryo showing the implantation site of the BMP5-soaked bead in E. (B) At stage 13, Wnt2b transcripts are restricted to the presumptive RPE (arrow). The arrowhead indicates Wnt2b expression in the ectoderm. (C) Wnt2b transcripts are detected in the RPE (arrows) and surface ectoderm (arrowhead) on the contralateral side of the BMP5-treated eye shown in E. (D) Wnt2b expression in the contralateral, untreated eye following implantation of noggin-expressing cells. Wnt2b transcripts are restricted to the RPE (arrowhead) and no transcripts are detected within the NR. The arrow shows Wnt2b expression within the ectoderm and anterior lens. (E) Following BMP5 application (asterisk), Wnt2b expression is also detected in the distal region of the optic vesicle, the presumptive NR (arrows). The arrowhead indicates Wnt2b expression in the ectoderm. (F) Parallel section of the noggin-treated eye shown in Fig. 5B,D. Wnt2b expression is downregulated in the entire outer optic cup (arrowheads). In the surface ectoderm (arrow) and anterior lens, Wnt2b expression is still detected.

Article Snippet: A 2 l drop of recombinant mouse BMP5 or BMP4 (0.7 mg/ml or 1 mg/ml; R&D Systems) was placed in a Petri dish and about eight drops (10 l each) of distilled water were placed around it to keep it from evaporating.

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Bone morphogenetic proteins specify the retinal pigment epithelium in the chick embryo.

doi: 10.1242/dev.02884

Figure Lengend Snippet: Fig. 4. Effects of BMP5 application on the distribution of genes expressed within the NR and RPE at optic cup stages. (A) Schematic illustrating the location of the BMP5-soaked bead following implantation at stage 10/11 as shown in E-I; B-D are PBS-soaked bead controls (B) In control embryos, Mitf expression is weakly detected in the RPE at stage 15 (arrowheads). (C) The RPE-specific marker MMP115 is restricted to the RPE at this stage (arrowheads). (D) Strong Rx expression is detected in the NR at this stage (arrowhead). (E) Following implantation of a BMP5-soaked bead (asterisk), optic cup formation is not observed and Mitf expression is detected in the distal optic vesicle (arrowheads). (F) Parallel section of the embryo shown in E and G. MMP115 expression is induced in the presumptive NR (arrowhead) and the optic stalk region following BMP5 exposure. (G) Implantation of a BMP5-soaked bead (asterisk) leads to downregulation of Rx expression in the presumptive NR. (H) BMP5 application downregulates Chx10 expression in the distal optic vesicle/cup, the presumptive NR (right, arrowheads). By contrast, Chx10 expression is strongly observed within the presumptive NR of the contralateral eye (left, arrow). (I) Parallel section of the embryo shown in H. MMP115 expression is induced by BMP5 in the presumptive NR (arrowheads), whereas in the contralateral, unoperated eye, MMP115 transcripts are absent from the NR (left, arrow). L, Lens.

Article Snippet: A 2 l drop of recombinant mouse BMP5 or BMP4 (0.7 mg/ml or 1 mg/ml; R&D Systems) was placed in a Petri dish and about eight drops (10 l each) of distilled water were placed around it to keep it from evaporating.

Techniques: Control, Expressing, Marker

Journal: JCI Insight

Article Title: Genomic analysis of benign prostatic hyperplasia implicates cellular relandscaping in disease pathogenesis

doi: 10.1172/jci.insight.129749

Figure Lengend Snippet: (A) Heatmap of genes with significant differential expression in BPH versus normal prostate. Samples are clustered (see color key) and genes ordered by SAM score (t statistic value). (B) Volcano plot (Q value vs. log2 fold change) annotated with topmost genes differentially expressed in BPH. (C) Technical validation of RNA-Seq results by qRT-PCR, confirming elevated expression of BMP5 and CXCL13 in BPH. Five BPH-normal pairs assayed once with technical quadruplicates. Graphed are mean (±1 SD) relative expression levels, normalized to GAPDH and compared against a reference sample (normal prostate sample 8791). Each data point represents 1 of up to 16 pairwise ratios (calculated from the quadruplicate test and reference values). Below, heatmap depiction of corresponding RNA-Seq (median-centered log2 reads per kilobase million) values.

Article Snippet: The 25,000–40,000 cells were plated in 6-well plates in duplicate, and then recombinant human BMP5 (R&D Systems), CXCL13 (R&D Systems), or vehicle control was added daily with media replacement.

Techniques: Quantitative Proteomics, Biomarker Discovery, RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: JCI Insight

Article Title: Genomic analysis of benign prostatic hyperplasia implicates cellular relandscaping in disease pathogenesis

doi: 10.1172/jci.insight.129749

Figure Lengend Snippet: (A) Addition of BMP5 (250 ng/ml) to RWPE-1 prostate epithelial and WPMY-1 myofibroblast cells activates the canonical SMAD pathway, demonstrated by 15- to 20-fold increased phospho-SMAD1/5/9 on Western blot. (B) BMP5 addition (concentrations indicated) to RWPE-1 cells increases cell numbers. Mean (red) and SD (blue) shown. Multiplicity-adjusted P values generated from 1-way ANOVA with post hoc comparison to no BMP5 control (Dunnett’s test); *P < 0.05. Data are representative of 3 independent experiments, each done with 2 samples assayed per concentration. (C) Addition of BMP5 (250 ng/ml) to RWPE-1 cells leads to dispersed cell growth (with fewer cell clusters) and to increased Transwell migration. Mean and SD shown; P value generated from 2-tailed Student’s t test. Data are representative of 2 independent experiments, each done with 3 samples assayed per condition. (D and E) BMP5 addition to RWPE-1 and WPMY-1 cells generates a transcriptional response significantly enriched for BPH (vs. normal prostate) overexpressed genes (left), and with WPMY-1 cells for BPH stromal signature genes (center), but not AR/secretory signature genes (right). Gene set enrichment analysis (GSEA) enrichment score P values are indicated.

Article Snippet: The 25,000–40,000 cells were plated in 6-well plates in duplicate, and then recombinant human BMP5 (R&D Systems), CXCL13 (R&D Systems), or vehicle control was added daily with media replacement.

Techniques: Western Blot, Generated, Comparison, Control, Concentration Assay, Migration

Journal: JCI Insight

Article Title: Genomic analysis of benign prostatic hyperplasia implicates cellular relandscaping in disease pathogenesis

doi: 10.1172/jci.insight.129749

Figure Lengend Snippet: (A) Two-dimensional projection (t-SNE plot) of single-cell transcriptomes stratifies prostate tissue cells (dots) into distinct clusters, identifiable by characteristic expression of marker genes, including (B) KRT13 (basal epithelium) (red intensity scales to maximum log2 expression), (C) KLK3 (secretory epithelium), and (D) DCN (fibroblasts). Additional cell type markers are shown in Supplemental Figure 6. (E) Normal prostate tissue cell subset, illustrating expression of (F) CHGA (neuroendocrine cells), (G) BMP5, and (H) CXCL13. (I) BPH tissue cell subset, illustrating expression of (J) CHGA, (K) BMP5, and (L) CXCL13. Note relative depletion in BPH tissue of CHGA-expressing neuroendocrine cells and enrichment of BMP5- or CXCL13-expressing myofibroblasts. Insets magnify select cell clusters. Inset magnification ×1.6.

Article Snippet: The 25,000–40,000 cells were plated in 6-well plates in duplicate, and then recombinant human BMP5 (R&D Systems), CXCL13 (R&D Systems), or vehicle control was added daily with media replacement.

Techniques: Expressing, Marker

Journal: International journal of biological sciences

Article Title: Inhibition of BRD4 Attenuates ER Stress-induced Renal Ischemic-Reperfusion Injury.

doi: 10.7150/ijbs.83040

Figure Lengend Snippet: Figure 1. Treatment with JQ1 ameliorates overexpression of UPR genes induced by thapsigargin and hypoxia in HK-2 cells. Renal TECs (HK-2 cell line) were treated with DMSO (Ctrl) or Tg (4 μM, 24 h) (A,B) or cultured under hypoxic conditions (1% O2, 5% CO2) at different times (t0, normoxia; t12, 12 h) (C,D). The inhibitor of BET proteins, JQ1(+), or its inactive enantiomer, JQ1(-), were co-cultured at the same indicated times under both cultured conditions. Gene expression levels of ATF4, XBP1 and ATF6 were analyzed by RT-PCR (A,C) and protein levels were analyzed by western blot (B,D). Data are expressed as the mean ± SEM of at least three independent experiments. GADPH and β-actin were used as housekeeping markers of RT-PCR and western blot, respectively. Statistical analyses involved use of the two-tailed Student’s paired t-test and the Wilcoxon test. *p<0.05 vs. control (DMSO) or t0 (normoxia) and # vs. cells treated with JQ1(-) + Tg or cells in hypoxia (t12) treated with JQ1(-).

Article Snippet: Membranes were incubated overnight at 4°C with the following primary antibodies: XBP1 spliced form (NBP2-20917, 1:1000; Novus Biologicals, Centennial, CO, USA), ATF4 (11815S, 1:1000; Cell Signaling Technology, Danvers, MA, USA), ATF6 (MAB71527-SP, 1:2000; R&D Systems, Minneapolis, MI, USA), BRD4 (A301-985A; Bethyl Laboratories, Montgomery, TX, USA), KIM-1 (AF1817, 1:1000; R&D Systems), NGAL (Sc-50350, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (4967S, 1:2000; Cell Signaling Technology); followed by incubation with HRP-conjugated IgG secondary antibody for 1 h. Immunostained bands were visualized using a chemiluminescence kit (Luminata Forte Western HRP Substrate; Merck Millipore), detected on an ImageLab system (Bio-Rad, Hercules, CA, USA), and quantified with ImageJ version 1.53c software (NIH Image).

Techniques: Over Expression, Cell Culture, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test, Control

Journal: International journal of biological sciences

Article Title: Inhibition of BRD4 Attenuates ER Stress-induced Renal Ischemic-Reperfusion Injury.

doi: 10.7150/ijbs.83040

Figure Lengend Snippet: Figure 2. Specific BRD4 silencing impairs expression of UPR genes induced under hypoxia. HK-2 cells were transfected with a specific siRNA against BRD4 (siRNA BRD4) or control siRNA (siRNA Ctrl, 40 nM, 48 h) before exposure to conditions of normoxia (t0) or hypoxia (t12). (A) Transcriptional and protein levels of BDR4 after specific silencing. Transcriptional levels of ATF4, XBP1 and ATF6 were determined by RT-PCR (B) and protein levels (C) assayed by western blot. Data are expressed as mean ± SEM of three independent experiments. Statistical analyses involved use of the two-tailed Student’s paired t-test and the Wilcoxon test. *p<0.05 vs. t0 (normoxia), # vs. siRNA Ctrl-treated cells.

Article Snippet: Membranes were incubated overnight at 4°C with the following primary antibodies: XBP1 spliced form (NBP2-20917, 1:1000; Novus Biologicals, Centennial, CO, USA), ATF4 (11815S, 1:1000; Cell Signaling Technology, Danvers, MA, USA), ATF6 (MAB71527-SP, 1:2000; R&D Systems, Minneapolis, MI, USA), BRD4 (A301-985A; Bethyl Laboratories, Montgomery, TX, USA), KIM-1 (AF1817, 1:1000; R&D Systems), NGAL (Sc-50350, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (4967S, 1:2000; Cell Signaling Technology); followed by incubation with HRP-conjugated IgG secondary antibody for 1 h. Immunostained bands were visualized using a chemiluminescence kit (Luminata Forte Western HRP Substrate; Merck Millipore), detected on an ImageLab system (Bio-Rad, Hercules, CA, USA), and quantified with ImageJ version 1.53c software (NIH Image).

Techniques: Expressing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test