Journal: Stem Cells International

Article Title: BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia

doi: 10.1155/2016/9536192

Figure Lengend Snippet: PCR primer sequences and product size.

Article Snippet: Recombinant rat GDNF and bFGF, recombinant human BMP4 factors, and mouse anti-BMP4 primary antibody were from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Stem Cells International

Article Title: BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia

doi: 10.1155/2016/9536192

Figure Lengend Snippet: Expression of BMP4 and its receptor subunits in testicular cells. (a) RT-PCR analyses of the expression of BMP4 and its receptors BMPR1A , BMPRII , ActRII , ActRIIB, and actin in isolated spermatocytes (Spc) and round spermatids (Spd), as well as in cultured spermatogonia (Spg) and Sertoli cells (Sertoli). RNA samples without RT but with PCR amplification were used as negative control. (b-c) Western blotting analysis of the expression of BMP4 and its receptors in 4-5-day-old mouse Spg, adult Spc and Spd, and Sertoli cells (b) and quantification of BMP4 protein in these cells (c). The premature and mature forms of BMP4 were labelled. (d-e) Western blotting analysis of BMP4 protein in mouse testis at indicated development stages (d) and quantification (e). (f) Western blotting analyses of secreted BMP4 in the medium from different cell cultures. Adult mouse testicular cells lysate (TC) was used as positive control. The data is presented as means ± SD from at least three independent experiments. Statistically significant differences among groups are indicated by asterisks, ∗ P < 0.05 or ∗∗ P < 0.01.

Article Snippet: Recombinant rat GDNF and bFGF, recombinant human BMP4 factors, and mouse anti-BMP4 primary antibody were from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Cell Culture, Amplification, Negative Control, Western Blot, Positive Control

Journal: Stem Cells International

Article Title: BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia

doi: 10.1155/2016/9536192

Figure Lengend Snippet: BMP4 protein is mainly expressed in mouse germ cells. (a) IFA analysed the localization of BMP4 and DAZL in 7-day mouse testis section (arrow head). (b) In adult mouse testis section, BMP4 was predominantly expressed in spermatocytes (arrow) and round spermatids (arrow head). (c-d) In testicular cells isolated from 4-5-day mice, BMP4 was predominantly expressed in germ cells (arrow) and, to a less extent, in Sertoli cells (arrow head). (e-f) BMP4 was also expressed in P3 (e) and P33 (f) cultured spermatogonia. Scale bar: 20 μ m.

Article Snippet: Recombinant rat GDNF and bFGF, recombinant human BMP4 factors, and mouse anti-BMP4 primary antibody were from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Cell Culture

Journal: Stem Cells International

Article Title: BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia

doi: 10.1155/2016/9536192

Figure Lengend Snippet: BMP4 activates Smad signalling pathway in cultured spermatogonia. (a-b) IFA analyses of spermatogonia cultured in serum-free medium using antibodies against phosphorylated Smad1, Smad5, and Smad8 in the presence of BMP4 (a) or control (b). (c-d) IFA analysis of spermatogonia cultured in 15% serum-containing medium in the presence of BMP4 (c) or control (d). (e-f) Western blotting analysis of phosphorylated (e) and total (f) Smad1/5/8 in spermatogonia under different culture conditions. (g) Relative total Smad1/5/8 level in each group in (f) after normalization to the internal control. Scale bar: 20 μ m.

Article Snippet: Recombinant rat GDNF and bFGF, recombinant human BMP4 factors, and mouse anti-BMP4 primary antibody were from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Western Blot

Journal: Stem Cells International

Article Title: BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia

doi: 10.1155/2016/9536192

Figure Lengend Snippet: BMP4 induces the expression of Stra8 and c-Kit cooperatively with RA in cultured spermatogonia. (a-b) Real-time PCR analysis of Stra8 expression in cultured spermatogonia pretreated with BMP4 or Noggin and followed by RA stimulation for 8 hours (a) or 24 hours (b). (c-d) Real-time PCR analysis of c-Kit expression in cultured spermatogonia pretreated with BMP4 or Noggin and followed by RA stimulation for 8 hours (c) or 24 hours (d). (e) Real-time PCR analysis of BMP4 mRNA expression in cultured spermatogonia followed by RA alone stimulation for 8 hours and 12 hours. The columns were labelled with A, B, C, and D, which means values are significantly different ( P < 0.05) among differently labelled groups; ∗ P < 0.05 or ∗∗ P < 0.01.

Article Snippet: Recombinant rat GDNF and bFGF, recombinant human BMP4 factors, and mouse anti-BMP4 primary antibody were from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Molecular cancer

Article Title: BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

doi: 10.1186/s12943-015-0441-y

Figure Lengend Snippet: Fig. 1 BMP4 is down-regulated in 344SQ_miR-200 cells. a The expression profile of immune-related genes in 344SQ_vector (vec) or 344SQ_miR-200 cells. Yellow: genes up-regulated by miR-200 overexpression; blue: genes down-regulated by miR-200 overexpression. b Quantitative RT-PCR (qRT-PCR) of Bmp4 and miR-200c in 13 murine lung adenocarcinoma cells. Cells were grouped into epithelial- or mesenchymal-like cells on the basis of the expression of EMT markers [13]. r and p, one-tailed Spearman’s rank correlation test. c qRT-PCR of Bmp4 in 393P, 344SQ, 344SQ_miR-200, 393P_Zeb1, and their control cells (vec). Expression levels were normalized to that of 393P (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. d BMP4 Western blot in 393P, 344SQ, 344SQ_vec, and 344SQ_miR-200 cells. Actin was used as a loading control. e RNA polymerase II (Pol-II) chromatin immunoprecipitation assay on the Bmp4 promoter region in 344SQ_vec and 344SQ_miR-200 cells. Bars denote DNA precipitation (% of input) from each sample. IgG was used as a negative control. Mean + SD, n = 3; p, two-tailed Student’s t-test

Article Snippet: Murine Bmp4 shRNAs (#TG516759) were purchased (Origene) and Myh10 shRNAs were cloned into the pLKO.1 vector (Addgene) according to the protocol (http:// www.addgene.org/tools/protocols/plko/).

Techniques: Expressing, Plasmid Preparation, Over Expression, Quantitative RT-PCR, One-tailed Test, Control, Two Tailed Test, Western Blot, Chromatin Immunoprecipitation, Negative Control

Journal: Molecular cancer

Article Title: BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

doi: 10.1186/s12943-015-0441-y

Figure Lengend Snippet: Fig. 2 GATA4 and GATA6, transcription factors of BMP4, are miR-200 target genes. a Luciferase assay of Bmp4 promoter activity. 393P cells were co-transfected with GATA4 or GATA6 expression vector and the Bmp4 promoter reporter. Results were normalized by using a Renilla luciferase vector (pRL-TK). Mean + SD, n = 3; p, two-tailed Student’s t-test. b, c Luciferase assay of Gata4 and Gata6 3′-UTR activity. 344SQ cells were co-transfected with control (con) or miR-200 mimics and 3′-UTR reporter constructs. Results were normalized by using a firefly luciferase vector (pGL3-con). ZEB1 3′-UTR construct was used as a positive control. Mean + SD, n = 3; p, two-tailed Student’s t-test. d qRT-PCR of Gata4, Gata6, and Zeb1 in 344SQ cells transfected transiently with control (con) or miR-200b mimic. The expression levels were normalized to those of the control group (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. e Bmp4 qRT-PCR in 344SQ_miR-200 cells transfected with GATA4 or GATA6. The expression levels were normalized to that of vector-transfected cells (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test

Article Snippet: Murine Bmp4 shRNAs (#TG516759) were purchased (Origene) and Myh10 shRNAs were cloned into the pLKO.1 vector (Addgene) according to the protocol (http:// www.addgene.org/tools/protocols/plko/).

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation, Two Tailed Test, Control, Construct, Positive Control, Quantitative RT-PCR

Journal: Molecular cancer

Article Title: BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

doi: 10.1186/s12943-015-0441-y

Figure Lengend Snippet: Fig. 3 BMP4 knockdown suppresses cancer cell growth, migration, invasion, tumorigenesis, and metastasis. a qRT-PCR of Bmp4 in 344SQ transfected with Bmp4 shRNAs (KD #2 and #3) or nontargeting control (NTC) vector. The expression levels were normalized to that of NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. b Western blot of BMP4, phospho-SMAD1/5 (pSMAD1/5), and total SMAD1/5 in 344SQ-Bmp4-KD cells. Actin was used as a loading control. (C) Cell numbers of 344SQ-Bmp4-KD and NTC cells over 7 days. Mean ± SD, n = 4; p, two-tailed Student’s t-test. (D) Diameters of 3-D acini of 344SQ-Bmp4-KD and NTC cells cultured on Matrigel for 10 days. Mean ± SD, n = 15; p, two-tailed Student’s t-test. e, f Migrated (e) and invaded (f) cells in Boyden chambers were photographed and counted. Mitomycin C (1 μg/mL) was added to the culture media to block cell proliferation. Mean + SD, n = 3; p, two-tailed Student’s t-test. g Primary tumor weight (left) and lung metastases (right) in syngeneic mice injected with 344SQ-Bmp4- KD and NTC cells. Box-and-whisker plots denote median and upper/lower quartiles + 1.5 × IQR (interquartile range). p, two-tailed Student’s t-test

Article Snippet: Murine Bmp4 shRNAs (#TG516759) were purchased (Origene) and Myh10 shRNAs were cloned into the pLKO.1 vector (Addgene) according to the protocol (http:// www.addgene.org/tools/protocols/plko/).

Techniques: Knockdown, Migration, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Expressing, Two Tailed Test, Western Blot, Cell Culture, Blocking Assay, Injection, Whisker Assay

Journal: Molecular cancer

Article Title: BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

doi: 10.1186/s12943-015-0441-y

Figure Lengend Snippet: Fig. 4 BMP4 is required for normal acinus formation in Matrigel 3-D culture. a, b Matrigel 3-D culture of 344SQ-NTC and Bmp4-KD cells (#2 and #3). Phase contrast (a) and confocal microscope (b) images of 3-D acini were taken 12 days after cell seeding. Blue: DAPI, red: β-catenin, green: ZO-1. c Matrigel 3-D culture of 344SQ_vec and miR-200 cells. Confocal images of 3-D acini were taken 12 days after cell seeding. Blue: DAPI, red: β-catenin, green: ZO-1. d Matrigel 3-D culture of 344SQ-NTC and Bmp4-KD cells (#2 and #3). Confocal images of 3-D acini were taken 7 days after cell seeding, and spindle angles of dividing cells were measured using ImageJ (http://imagej.nih.gov/ij/). Blue: DAPI, red: α-tubulin. White arrow: direction of cell division; yellow arrow with asterisk: spindle angle. Box-and-whisker plot denotes median and upper/lower quartiles + 1.5 × IQR. p, two-tailed Student’s t-test

Article Snippet: Murine Bmp4 shRNAs (#TG516759) were purchased (Origene) and Myh10 shRNAs were cloned into the pLKO.1 vector (Addgene) according to the protocol (http:// www.addgene.org/tools/protocols/plko/).

Techniques: Microscopy, Whisker Assay, Two Tailed Test

Journal: Molecular cancer

Article Title: BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

doi: 10.1186/s12943-015-0441-y

Figure Lengend Snippet: Fig. 5 Non-muscle myosin is a downstream target of BMP4. a Heatmap of differentially expressed genes (fold change ≥1.5, p value <0.05) identified by mRNA microarray of 344SQ-NTC and Bmp4-KD (#2) cells. Yellow: increased expression; blue: decreased expression. b qRT-PCR of 29 selected genes to confirm the microarray data. Mean + SD, n = 3; p, two-tailed Student’s t-test. c qRT-PCR of Bmp4 and Myh10 in 344SQ-NTC and Bmp4-KD cells (#2 and #3). The expression levels were normalized to those of 344SQ-NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. d qRT-PCR of Myh10 in 344SQ-NTC and Myh10-KD cells (#3 and #4). The expression levels were normalized to those of 344SQ-NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. e, f Matrigel 3-D culture of 344SQ-NTC and Myh10-KD cells (KD#3 and #4; E), and 344SQ cells in the presence or absence of blebbistatin (Bleb, 25 μM; F). Phase contrast microscope images of 3-D acini were taken 12 days after cell seeding

Article Snippet: Murine Bmp4 shRNAs (#TG516759) were purchased (Origene) and Myh10 shRNAs were cloned into the pLKO.1 vector (Addgene) according to the protocol (http:// www.addgene.org/tools/protocols/plko/).

Techniques: Microarray, Expressing, Quantitative RT-PCR, Two Tailed Test, Microscopy

Journal: Molecular cancer

Article Title: BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

doi: 10.1186/s12943-015-0441-y

Figure Lengend Snippet: Fig. 6 JAG2, miR-200, and BMP4 form a regulatory loop. a qRT-PCR of Jag1, Jag2, and Bmp4 in 344SQ-NTC and Bmp4-KD cells (#2 and #3). The expression levels were normalized to those of 344SQ-NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. b qRT-PCR of Jag1, Jag2, and Bmp4 in 344SQ-Bmp4- KD cells transfected with BMP4 expression vector and control (vec). The expression levels were normalized to those of vector-transfected groups (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. c qRT-PCR of Jag1, Jag2, and Bmp4 in 393P cells transfected with BMP4 expression vector and control (vec). The expression levels were normalized to those of 393P-vec (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. d Jag2 qRT-PCR in 393P cells treated with BMP4 protein. The expression levels were normalized to that of non-treated control (=1.0). Mean + SD, n = 3; *p < 0.01, two-tailed Student’s t-test. e, f Luciferase assay of JAG2 promoter activity in 393P cells treated with BMP4 protein (E) or co-transfected with SMAD1 and/or SMAD5 expression vectors (f). Results were normalized by using a Renilla luciferase vector (pRL-TK). Mean + SD, n = 3; p, two-tailed Student’s t-test. g qRT-PCR of Jag1, Jag2, Bmp4, and Gata3 in 344SQ cells transfected with a Jag2 shRNA (KD) or nontargeting control (NTC) vector. The expression levels were normalized to those of 344SQ-NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. h qRT-PCR of miR-200 family members (200a, 200b, 200c, 141, and 429) in 344SQ-Jag2- KD and 344SQ-NTC. The expression levels were normalized to those of 344SQ-NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. i qRT-PCR of miR-200 family members in 344SQ-NTC and Bmp4-KD (#2 and #3) cells. The expression levels were normalized to those of 344SQ-NTC (=1.0). Mean + SD, n = 3; p, two-tailed Student’s t-test. j Diagram of a regulatory loop involving BMP4, JAG2, and miR-200

Article Snippet: Murine Bmp4 shRNAs (#TG516759) were purchased (Origene) and Myh10 shRNAs were cloned into the pLKO.1 vector (Addgene) according to the protocol (http:// www.addgene.org/tools/protocols/plko/).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, shRNA

Journal: Cytokine

Article Title: Bmp4 expressed in preadipocytes is required for the onset of adipocyte differentiation.

doi: 10.1016/j.cyto.2013.07.011

Figure Lengend Snippet: Fig. 3. Endogenous Bmp activity prior to differentiation induction is required for efficient lipid accumulation in 3T3-L1 cells. (A) Smad phosphorylation in response to the Tgf-b family in 3T3-L1 cells treated with dorsomorphin or LDN-193189. 3T3- L1 cells at 70% confluence were pre-treated with dorsomorphin (DM: 5 lM), LDN- 193189 (LDN: 100 nM) or DMSO as a vehicle control (V) for 20 min, followed by treatment with Tgf-b1 (200 pM), Activin A (4 nM) or Bmp2 (4 nM) for 1 h. Phosphorylated Smad1/5/8 (upper panel) and Smad2 (third panel) were examined by Western blotting, and the membranes were then re-blotted for total Smad1 (second panel) and Smad2 (fourth panel). (B) Effects of dorsomorphin and LDN-193189 on Bmp-mediated transcription. 3T3-L1 cells were transiently transfected with BRE- luc and Renilla-luc and treated with dorsomorphin (DM: 5 lM), LDN-193189 (LDN: 100 nM) or DMSO as a vehicle control (V) for 20 min, followed by treatment with or without Bmp4 (4 nM) for 16 h. Firefly luciferase activity was normalized to Renilla luciferase activity, and the luciferase activity in cell lysates in the absence of Bmp4 and inhibitors was set to 1. Data were expressed as the mean ± SE of triplicates from a representative experiment. (C) Suppression of lipid accumulation by the inhibi- tion of Bmp activity in 3T3-L1 cells. Dorsomorphin (DM: 5 lM), LDN-193189 (LDN: 100 nM) or DMSO as a vehicle control (V) was added to the culture medium from day 2 to day 0, and lipid accumulation on day 8 was examined by Oil Red O staining (upper panel). After staining, the dye was extracted with 2-propanol, and the amount was quantified by measuring the absorbance at 510 nm. The absorbance of the extracted dye from cells treated with vehicle was set to 100. Data are expressed as the mean ± SE of quadruplicates. P < 0.01 vs. vehicle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following reagents were purchased: dorsomorphin (compound C: 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine) was from Calbiochem (La Jolla, CA, USA);LDN-193189 was from Stemgent (San Diego, CA, USA);purified Tgf-b1 was from Becton Dickinson (Franklin Lakes, NJ, USA);recombinant Activin A, Bmp2 and Bmp4 were from R & D Systems (Minneapolis, MN, USA);rabbit polyclonal antibody against phospho-Smad1 (Ser463/Ser465)/Smad5 (Ser463/Ser465)/Smad8 (Ser426/Ser428) (#9511) and rabbit polyclonal antibody against phospho-Smad2 (Ser465/Ser467) were from Cell Signaling Technology (Danvers, MA, USA);and rabbit monoclonal antibody against Smad1 (ab33902), rabbit polyclonal antibody against Smad2 (ab63576), and mouse monoclonal antibody against and b-actin (AC-15) were from Abcam (Cambridge, MA, USA).

Techniques: Activity Assay, Phospho-proteomics, Control, Western Blot, Transfection, Luciferase, Staining

Journal: Scientific Reports

Article Title: A novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering

doi: 10.1038/srep04457

Figure Lengend Snippet: Release of BMP4 and CBD-BMP4 from gelatin-FA hydrogels

Article Snippet: Recombinant mouse BMP4, affinity purified anti-mouse BMP4 polyclonal IgG, and biotinylated anti-mouse BMP4 IgG were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Scientific Reports

Article Title: A novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering

doi: 10.1038/srep04457

Figure Lengend Snippet: BMP4 or CBD-BMP4 (each 250 nM) was mixed into 15% gelatin-FA containing 0.05% RB, and the mixtures were implanted into osteochondral bone defects and exposed to visible light for 2 min. (a) Representative photographs of the gross appearance after the procedure (five femurs each). (b) Gross grading scores at 4 and 12 weeks after the procedure (five femurs each). (c) Representative images of safranin O staining (five femurs each). The scale bar indicates 500 μm. (d) Histological grading scores at 4 and 12 weeks (five femurs each). (e) The expression levels of chondrogenic factors at 4 weeks (5 femurs each). * P < 0.05, ** P < 0.01, *** P < 0.001 (five femurs each). **** P < 0.05, vs. untreated control (five femurs each, Mann–Whitney U Test).

Article Snippet: Recombinant mouse BMP4, affinity purified anti-mouse BMP4 polyclonal IgG, and biotinylated anti-mouse BMP4 IgG were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Expressing, Control, MANN-WHITNEY

Journal: Methods in Molecular Biology

Article Title: Molecular Dermatology

doi: 10.1007/978-1-62703-227-8

Figure Lengend Snippet: Fig. 2. The stages of iPSC differentiation during keratinocyte derivation. iPSCs generated from an ICR mouse were differ- entiated into keratinocytes using differentiation Protocol I. ( a ) EBs treated with RA for 2 days in suspension culture. ( b ) EB outgrown on a ColIV-coated plate in the presence of BMP4 on day 10 of differentiation. ( c ) EB outgrown on a ColIV-coated plate on day 17 of differentiation before rapid attachment plating. ( d ) iPSC-derived keratinocytes at passage 2 post-rapid attachment plating (day 25 of differentiation). All images were taken with 10× objectives.

Article Snippet: 100 ng/ m l stock solution of mouse BMP4 (R&D Systems) reconstituted in sterile 4 mM HCl containing 0.1% bovine serum albumin (BSA).

Techniques: Generated, Suspension, Derivative Assay