Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: G9a knockdown may lead to increase in BMP5 expression. ( A ) Regulatory factors suppressing tumor aggressiveness were identified by using microarray analysis. ( B ) Expression levels for each gene were compared (ratios between gene expression levels of non-silencing control). In two G9a knockdown cells (shG9a #1 and shG9a #2), expression levels for each gene were compared to those of non-silencing control. Then the most upregulated and downregulated genes were screened. ( C ) G9a knockdown cells showed increase in BMP5 expression. ( D ) Reduced G9a occupancy was found at the BMP5 promoter region. G9a knockdown cells also showed decreased H3K9me2 occupancy at the promoter region of BMP5. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Knockdown, Expressing, Microarray, Gene Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: BMP5 contributes to reduce migration and invasion abilities of tumor cells. ( A ) Low BMP5 expression is associated with poor survival outcomes of breast cancer patients. ( B ) Patients with stage 3 disease showed higher BMP5 levels than those of stage 2 patients. ( C , D ) Migration/invasion abilities were decreased by treatment of recombinant BMP5; however, the capabilities were enhanced following BMP5 downregulation. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Migration, Expressing, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: G9a knockdown facilitates Smad protein phosphorylation via BMP5 activation. ( A , B ) G9a-knockdown-induced increase in BMP5 expression had no effect on the total level of either Smad1 or Smad5. G9a knockdown increased phosphorylation of Smad1/5/9. ( C ) ICC demonstrated that nuclear translocation of pSmad1/5/9 was increased in G9a-depleted MCF7 cells. ( D ) Similar tendency was found after treatment of recombinant BMP5. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Knockdown, Phospho-proteomics, Activation Assay, Expressing, Translocation Assay, Recombinant

Journal: Nature Communications

Article Title: Bone marrow-derived epithelial cells and hair follicle stem cells contribute to development of chronic cutaneous neoplasms

doi: 10.1038/s41467-018-07688-8

Figure Lengend Snippet: CD34 − , CD44 + BMCs express keratin after BMC/KC co-culture and BMP5 treatment. a , b All adherent BMCs are CD34-negative and CD44-positive. c , e A sub set of adherent BMCs are pan-keratin-immunoreactive (arrowheads) after 7days of BMC/KC co-culture (keratin-positive BMCs in white boxes are magnified and merged with phase image). d Pan-keratin-immunoreactive BMC (arrowhead) identified 10 days after BMP5 treatment. f K14-immunoreactive cells (arrowheads) 10 days after BMP5 treatment (white box area is magnified). g Histogram of number of keratin-expressing BMCs; BMCs without treatment, BMC/KC co-culture (pan-keratin-positive BMCs, gray bar) and BMP5 treatment (K14-positive BMCs, black bar), pan-keratin- and K14-immunoreactive BMCs are detected in KC co-cultured and BMP5-treated BMCs, but no keratin-positive cells are detected in treatment controls ( n = 3, 3 different culture groups, 3 male and 3 female mice in each group; P < 0.017 as determined by Student’s t -test, mean ± s.d.) h Q-RT-PCR results show the relative level of K14 expression detected from positive (primary KCs and Tg.AC, a KC cancer cell-line) and negative (Swiss mouse 3T3 cell-line and primary BMCs) control groups, and experimental (BMC/KC co-culture and BMP5 treatment) groups. The expression levels were normalized to GAPDH expression levels, and expression mean values were converted into fold-change gene expression. Final values were normalized with log 2 transformation. ( n = 3, P < 4.81 × 10E−12 as determined by Fisher’s ANOVA method, mean ± s.d.). *White scale bar, 50 µm; Red scale bar, 200 µm

Article Snippet: For BMP5 (R&D systems, Minneapolis, MN; Cat# 6176-BM/CF) treatment, mesenchymal stem cell medium was changed every three days for 10 days, and BMP5 (50 ng/ml) was added immediately after changing medium (Supplementary Figure ).

Techniques: Co-Culture Assay, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control, Gene Expression, Transformation Assay

Journal: Cancers

Article Title: Exploring Spatial Heterogeneity of Immune Cells in Nasopharyngeal Cancer.

doi: 10.3390/cancers15072165

Figure Lengend Snippet: Figure 1. Differential expression profiles in CD45+ segments from ‘immune-rich cancer cell islets’ and ‘surrounding stromal leukocyte’ regions in NPC. (a) AOIs were generated based on cell expression of the morphological markers CD45 (red), PanCK (cyan), and CD8 (magenta) from the ROIs, and the collected oligonucleotides were quantified using the GeoMx® DSP system. (b) Biplot principal component analysis showing ROI segments (AOIs) as well as protein markers on the first and second components from ‘immune-rich cancer cell islets’ and ‘surrounding stromal leukocyte’ regions. (c) Heatmap demonstrating DE proteins between ‘immune-rich cancer cell islets’ and ‘surrounding stromal leukocyte’ regions (Mann–Whitney test, p-value < 0.05). (d) Twenty DE proteins’ correlation map (Pearson’s correlation, significance taken at |r| > 0.7, p-value < 0.05).

Article Snippet: Glass slides containing 5 μm sections of the FFPE TMAs were stained with DNA stain (SYTOTM 13 Green Fluorescent Nucleic Acid Stain, Invitrogen, Waltham, MA, USA), fluorescently labelled antibodies against CD8 (CD8-AF666/Cy5, clone OTI3H6, Origene, Rockville, MD, USA), PanCK (pan-Cytokeratin-AF568/Cy3, clone AE1 + AE3, Novus Biologicals, Littleton, CO, USA), and CD45 (CD45-AF615/Texas Red, clone 2B11 + PD7/26, Novus Biologicals, Eaglewood, CO, USA) (Figure S1 and Table 1).

Techniques: Quantitative Proteomics, Generated, Expressing, MANN-WHITNEY

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 1. Specificity of the anti-BMP5 antibody. Wells with BMP5 antibody show intense ELISA reaction only when coated by BMP5 protein and not coated by BMP6 and BMP7 protein. Wells without the first antibody (control) show no ELISA reaction (A). Western blot analysis using anti-BMP5 antibody for the whole rat brain shows a single band of about 17 kDa (B).

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 2. BMP5 expression in the whole rat brain (A1, B1, C1, D1, E1, F1, G1, H1). Control sections without the primary antibody (A2, B2, C2, D2, E2, F2, G2, H2). 12, hypoglossal nucleus; Amy, amygdala; CN, cerebellar nuclei; Cor, cerebral cortex; CPu, caudate putamen; DH, dorsal horn; Hi, hippocampus; IO, inferior olive; MG, medial geniculate; MV, medial vestibular nucleus; OB, olfactory bulb; Sol, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; Th, thalamus, VH, ventral horn. Scale bar = 0.5 mm for A, G, H; 1 mm for B–F.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Control

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 4. BMP5 expression in the olfactory bulb (A–D), medial septal nucleus (E), lateral septal nucleus (F), ventral pallidum (G), islands of Calleja (H) and piriform cortex (I). Note that the pia mater (arrows in D), perivascular space (arrowheads in D), and ependyma of the lateral ventricle (arrowheads in F) were very strongly stained. In the olfactory bulb, the soma of mitral cells (arrowheads in C) was strongly stained, and periglomerular neurons (arrowheads in B) were moderately stained, and glomeruli (asterisks in B) were weakly stained. In the ventral pallidum, the somas (arrowheads in G) and the dendrites (arrow in G) of neurons were strongly stained. In the piriform cortex, pyramidal neurons (arrowheads in I) were moderately stained. I–III, layers I–III of the piriform cortex; DB, diagonal band; EPI, external plexiform layer; Gl, glomerular layer; Gr, granular layer; ICj, islands of Calleja; LS, lateral septal nucleus; Mi, mitral cell layer; MS, medial septal nucleus; ON, olfactory nerve layer; Pir, piriform cortex; SE, subependymal layer; VP, ventral pallidum. Scale bar = 400 lm for E; 160 lm for A, I; 80 lm for F; 40 lm for B–D, G, H.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 5. BMP5 expression in the cerebral cortex (A–C), hippocampus (D–F) and corpus callosum (G). In the cerebral cortex, the pia mater (arrows in A) was very strongly stained, and the apical dendrites (arrows in B and C) and the somas (arrows in C) of pyramidal cells in the layer V, and neuropil in the layer I (asterisks in B) were strongly stained. In the hippocampus, the apical dendrites of pyramidal cells (arrows in E) were strongly stained, and pyramidal cells of the Ammon’s horn (arrowheads in E) were moderately stained, and weak BMP5-IR-positive cells were observed in the corpus callosum (arrowheads in G). I–VI, layers I–VI; cc, corpus callosum; CA1, 3, field CA1, 3 of Ammon’s horn; DG, dentate gyrus; Gr, granular layer; Mo, stratum molecular; Or, stratum oriens; Po, polymorphological layer; Py, stratum pyramidale; Ra, stratum radiatum. Scale bar = 400 lm for D; 160 lm for A; 80 lm for F; 40 lm for B, C, E, G.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 6. BMP5 expression in the basal ganglia (A, B), thalamus (C–G) and hypothalamus (H–K). Note that the pia mater (arrows in C), perivascular space (arrowheads in E and F), and the ependyma of the third ventricle (arrowheads in K) were very strongly stained. CPu, caudate putamen; GP, globus pallidus; LG, lateral geniculate nucleus; LHb, lateral habenular nucleus; MHb, medial habenular nucleus; Po, posterior thalamic nucleus; PV, paraventricular hypothalamic nucleus; Rt, reticular thalamic nucleus; SO, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus; VP, ventroposterior thalamic nucleus. Scale bar = 400 lm for C; 160 lm for G, I, K; 80 lm for A, B, D, H, J; 40 lm for E, F.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 7. BMP5 expression in the midbrain (A–C), motor system (D–G) and general somatosensory system (H–J). Note that the pia mater (arrows in A) and the ependyma (arrowheads in A) were very strongly stained, that neurons in the red nucleus (arrowheads in B) and the axons in the facial nerve (arrows in F) were strongly stained, and that neurons in the pars compacta of the substantia nigra (arrowheads in C) and the trigeminal mesencephalic nucleus (arrowheads in H) were moderately stained. 5, motor trigeminal nucleus; 7, facial nucleus; 7n, facial nerve; 12, hypoglossal nucleus; CG, central gray; Cu, Cuneate nucleus; Gr, Gracile nucleus; IP, interpeduncular nucleus; Me5, trigeminal mesencephalic nucleus; MG, medial gray; R, red nucleus; SN, substantia nigra; Sp5, trigeminal spinal nucleus. Scale bar = 400 lm for A; 80 lm for H, J; 40 lm for B–G, I.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 8. BMP5 expression in the general viscerosensory system (A, B) and special somatosensory system (C–G). Note very strong BMP5-IR in the nucleus of the solitary tract and dorsal nucleus of the vagus (A, B). Note that the ependyma of the fourth ventricle (arrowheads in A and D) and the pia mater (arrows in G) were very strongly stained, and that axons in the vestibulocochlear nerve (arrows in F) were strongly stained. 8n, vestibulocochlear nerve; 10, vagus nucleus; 12, hypoglossal nucleus; DC, dorsal cochlear nucleus; LVe, lateral vestibular nucleus; MVe, medial vestibular nucleus; SC, superior colliculus; Sol, nucleus of the solitary tract; VC, ventral cochlear nucleus. Scale bar = 160 lm for A, C–E, G; 80 lm for B; 40 lm for F.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 9. BMP5 expression in the other lower brain stem areas (A–G). Note that the locus coeruleus (D, E) and the ependyma of the fourth ventricle (arrowheads in A and D) show very strong BMP5-IR. In the gigantocellular reticular nucleus, the somas (arrowheads in F) and the dendrites (arrows in F) of neurons were very strongly stained. IO, inferior olive; LC, locus coeruleus; Me5, mesencephalic trigeminal nucleus; PDTg, pontine dorsal tegmental nucleus; Pn, pontine nucleus; Rt, reticular nucleus. Scale bar = 160 lm for A, D; 80 lm for G; 40 lm for B, C, E, F.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 10. BMP5 expression in the cerebellum (A, B), spinal cord (C–G), subventricular zone (H) and choroidal plexus (I). Note that axons (arrows in D) and astrocyte-like cells (arrowheads in D) in the white matter and the somas (arrowheads in G) and the dendrites (arrows in G) of motor neurons in the ventral horn were strongly stained. Note very strong BMP-IR in the Purkinje layer (A) and layers I and II of the dorsal horn of the spinal cord (F). I–III, layers I–III of the spinal cord; Ch, choroidal plexus; CN, cerebellar nuclei; DH, dorsal horn; Gr, granular layer; Mol, molecular layer; Pur, Purkinje cell layer; SVZ, subventricular zone; VH, ventral horn; wm, white matter. Scale bar = 400 lm for C; 160 lm for B, E; 80 lm for F, I; 40 lm for A, G, H; 16 lm for D.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: BMP5 expression in the adult rat brain.

doi: 10.1016/j.neuroscience.2014.07.057

Figure Lengend Snippet: Fig. 11. Double staining study showing that BMP5-IR-positive cells in the mitral cells (A1) are also positive for Tbx21 (A2), BMP5-IR-positive cells in the granular cells (B1), caudate putamen (C1), reticular thalamic nucleus (D1), cerebral cortex (E1) and hippocampus (F1) are also positive for NeuN (B2–F2), BMP5-IR-positive cells in the corpus callosum (G1) and hippocampus (H1) are also positive for GFAP (G2, H2), BMP5-IR-positive axons in the facial nerve (I1) and vestibulocochlear nerve (J1) are also positive for pan-neuronal marker (I2, J2) in a merged photomicrograph (A3–J3). cc, corpus callosum; Cor, cerebral cortex; CPu, caudate putamen; Gr, granular cell; Hip, hippocampus; Mi, mitral cell; Rt, reticular thalamic nucleus; 7n, facial nerve; 8n, vestibulocochlear nerve. Scale bar = 20 lm.

Article Snippet: After the plate was washed three times with 0.01 M PBS, 50 ll of anti-BMP5 antibody (diluted 1:100; the final concentration, 1.0 lg/ ml; R&D Systems, Inc.) was added to each well and then incubated for 2 h at RT.

Techniques: Double Staining, Marker