Journal: Hypertension

Article Title: SMAD1 Deficiency in Either Endothelial or Smooth Muscle Cells Can Predispose Mice to Pulmonary Hypertension

doi: 10.1161/hypertensionaha.111.199158

Figure Lengend Snippet: Figure 3. Impaired response to bone morphogenetic protein 4 (BMP4) but not to BMP7 in Bmpr2-deleted pulmonary endothelial cells (pECs). A, Western blot shows undetectable level of bone morphogenetic protein receptor type 2 (BMPR2) protein in pECs (1f/1f). Smad1,5,8 phosphorylation was increased in a dose-dependent manner at various concentrations (0–50 ng/ mL) of BMP4 treatment for 30 minutes after serum starvation on Bmpr2-intact (Bmpr22f/2f) cells. The level of pSMAD1,5,8 by BMP4 was markedly reduced in Bmpr2-deleted pECs (Bmpr21f/1f) at all dosages. B, Phosphorylation of SMAD1/5/8 by BMP7 (0–50 ng/mL) was unaffected in Bmpr21f/1f cells. C and D, Statistical analysis of the Western blot data shown in A and B, respectively. pSMAD1,5,8/SMAD1/β-actin ratio was increased in a dose-dependent manner by BMP4 in Bmpr22f/2f cells but not in Bmpr21f/1f cells (C). The levels of pSMAD1/5/8 were increased by BMP7 in both Bmpr22f/2f and Bmpr21f/1f cells. r2 and P values in regression analysis are indicated. Mean and standard error of data obtained from triplicate samples are shown at each data point.

Article Snippet: Recombinant mouse TGF-β1, BMP4, and BMP7 were purchased from R&D Systems.

Techniques: Western Blot, Phospho-proteomics

Journal: Hypertension

Article Title: SMAD1 Deficiency in Either Endothelial or Smooth Muscle Cells Can Predispose Mice to Pulmonary Hypertension

doi: 10.1161/hypertensionaha.111.199158

Figure Lengend Snippet: Figure 5. Bone morphogenetic protein (BMP) and transforming growth factor- β (TGF-β) signalings form an opposing balance in pulmonary endothelial cells (pECs). A, Western blot analysis shows that SMAD2 phosphorylation by TGF-β1 (0–2 ng/mL) is inhibited by BMP4 (25 ng/ mL) in BMPR2-intact 2f/2f cells, but this inhibitory effect of BMP4 is blunted in BMPR2-deficient 1f/1f cells. B, Inhibitory effect of BMP7 (25 ng/mL) on SMAD2 phosphorylation by TGF-β1 is unaffected in BMPR2-deficient pECs. Error bars are SEM (**P<0.01 and ***P<0.001).

Article Snippet: Recombinant mouse TGF-β1, BMP4, and BMP7 were purchased from R&D Systems.

Techniques: Western Blot, Phospho-proteomics

Journal: Neuroscience research

Article Title: The effects of Bone Morphogenetic Protein 4 on adult neural stem cell proliferation, differentiation and survival in an in vitro model of ischemic stroke.

doi: 10.1016/j.neures.2022.07.004

Figure Lengend Snippet: Fig. 2. Inhibitory effects of BMP4 on the proliferation of adult NSCs. NSC derived from the SVZ were cultured under normoxic conditions and three different concentrations (10, 20 and 40 ng/ml) of BMP4 were tested. (A) Immunocytochemistry images of adult NSCs. The proliferation marker: Ki67 (Red), neural stem cells marker: Nestin (Green) and DAPI were used. For all images, scale bar = 50 µm. (B) Bar graph of cell counts showing the percentage of Ki67-positive cells in different BMP4 concentrations. Data are represented as Mean ± SEM (n = 4 per group). One-way analysis of variance (ANOVA) followed by Tukey multiple comparison test was performed to compare the mean values. *p < 0.05; **p < 0.01; and *** p < 0.001 were considered statistically significant.

Article Snippet: Recombinant BMP4 (R&D Systems, 5020-BP) was added into media of treatment groups 48 h post seeding.

Techniques: Derivative Assay, Cell Culture, Immunocytochemistry, Marker, Comparison

Journal: Neuroscience research

Article Title: The effects of Bone Morphogenetic Protein 4 on adult neural stem cell proliferation, differentiation and survival in an in vitro model of ischemic stroke.

doi: 10.1016/j.neures.2022.07.004

Figure Lengend Snippet: Fig. 3. Effects of BMP4 on the differentiation of adult NSCs. Adult NSCs were cultured under normoxic conditions for differentiation assays. (A) Immunocyto chemistry images of adult NSCs that differentiated into β-III tubulin-positive neurons (Green). (B) Bar graph of cell counts showing the percentage of β-III tubulin- positive cells in the control and BMP4 treatment groups. (C) Immunocytochemistry images of adult NSCs that differentiated into GFAP-positive astrocytes (Red). (D) Bar graph of cell counts showing the percentage of GFAP-positive astrocytes in the control and BMP4 treatment groups. (E) Immunocytochemistry images of adult NSCs that differentiated into AQP4-positive astrocytes (Red). (F) Bar graph of cell counts showing the percentage of AQP4-positive astrocytes in the control and BMP4 treatment groups. For (B) and (D) and (F), data are expressed as Mean ± SEM (n = 4 per group). Student’s t-test was performed to compare means. *p < 0.05; **p < 0.01; and ***p < 0.001 were considered statistically significant. Scale bar = 50 µm, is representative of all images in A, C and E.

Article Snippet: Recombinant BMP4 (R&D Systems, 5020-BP) was added into media of treatment groups 48 h post seeding.

Techniques: Cell Culture, Control, Immunocytochemistry

Journal: Neuroscience research

Article Title: The effects of Bone Morphogenetic Protein 4 on adult neural stem cell proliferation, differentiation and survival in an in vitro model of ischemic stroke.

doi: 10.1016/j.neures.2022.07.004

Figure Lengend Snippet: Fig. 5. Inhibitory effects of BMP4 treatment on the proliferation of adult NSCs persist in OGD ischemic conditions. (A) Immunostaining of adult NSCs for the proliferation marker, Ki67 (Red) in control, hypoxic and anoxic conditions, in the presence or absence of BMP4 (10 ng/ml). Scale bar = 50 µm (B) Bar graph of cell counts showing the percentage of Ki67-positive cells to DAPI-positive cells among the groups. OGD resulted in a significant increase in proliferating (Ki67-positive) cells compared to the control group. Moreover, BMP4 exerted a strong inhibitory effect on adult NSC to result in a significant drop in the proliferative response in both OGD (hypoxia and anoxia) groups. Data are expressed as Mean ± SEM (n = 4). Two-way ANOVA followed by Tukey multiple comparison test was performed to compare means. *p < 0.05; **p < 0.01; and ***p < 0.001 were considered statistically significant.

Article Snippet: Recombinant BMP4 (R&D Systems, 5020-BP) was added into media of treatment groups 48 h post seeding.

Techniques: Immunostaining, Marker, Control, Comparison

Journal: Neuroscience research

Article Title: The effects of Bone Morphogenetic Protein 4 on adult neural stem cell proliferation, differentiation and survival in an in vitro model of ischemic stroke.

doi: 10.1016/j.neures.2022.07.004

Figure Lengend Snippet: Fig. 6. Effects of BMP4 treatment on the differentiation of adult NSCs in ischemic conditions. (A) Immunostaining of adult NSCs for the neuronal marker, β-III tubulin (Green). (B) Bar graph of cell counts showing the percentage of β-III tubulin-positive cells to DAPI-positive cells among the groups. β-III tubulin-positive neurons were significantly increased in the 0 % OGD group compared to the control group. Additionally, BMP4 treatment in both OGD (hypoxia and anoxia) groups was associated with a reduction in β-III tubulin-positive neurons. Immunostaining for (C) GFAP (Red) and (D) AQP4 (Red), the astrocytic markers, in control, hypoxic and anoxic conditions, in the presence or absence of BMP4 (10 ng/ml). (E) Bar graph of GFAP-positive cell counts. GFAP-positive cells were also significantly increased in OGD (hypoxia/anoxia) groups compared to the control group. Furthermore, BMP4 (10 ng/ml) potentiated the adult NSCs response to increase astrocytic differentiation in both OGD (hypoxia and anoxia) groups. (F) Bar graph of AQP4-positive cell counts. AQP4-positive cells did not display an increase in cell count under OGD conditions. However, BMP4 treatment resulted in significant increase in AQP4-positive cell count under normoxia and OGD conditions. For all images, scale bar = 50 µm. Data are expressed as Mean ± SEM (n = 4). Two-way ANOVA followed by Tukey multiple comparison test was performed to compare means. *p < 0.05; **p < 0.01; and ***p < 0.001 were considered statistically significant.

Article Snippet: Recombinant BMP4 (R&D Systems, 5020-BP) was added into media of treatment groups 48 h post seeding.

Techniques: Immunostaining, Marker, Control, Cell Counting, Comparison

Journal: Neuroscience research

Article Title: The effects of Bone Morphogenetic Protein 4 on adult neural stem cell proliferation, differentiation and survival in an in vitro model of ischemic stroke.

doi: 10.1016/j.neures.2022.07.004

Figure Lengend Snippet: Fig. 7. Id genes expression patterns in response to in vitro ischemia and/or BMP4 treatment. Expression of Id1 (A), Id2 (B), Id3 (C) and Id4 (D) mRNA quantified by qPCR in adult NSCs cultures exposed to normoxic (control) and OGD conditions, with or without BMP4 treatment. Analysis of Id genes expression displayed as fold changes of control group. mRNA expression is normalized to β-Actin (internal reference gene). Data are expressed as Mean ± SEM (n = 4). Two-way ANOVA followed by Tukey multiple comparison test was performed to compare means. *p < 0.05; **p < 0.01; and ***p < 0.001 vs control group, #p < 0.05; # #p < 0.01; and # # #p < 0.001 for 2 % OGD vs 2 % OGD + BMP4 group, ‡p < 0.05; ‡‡p < 0.01; and ‡‡‡p < 0.001 for 0 % OGD vs 0 % OGD + BMP4 group were considered statisti cally significant.

Article Snippet: Recombinant BMP4 (R&D Systems, 5020-BP) was added into media of treatment groups 48 h post seeding.

Techniques: Expressing, In Vitro, Control, Comparison

Journal: PloS one

Article Title: Overexpression of MyrAkt1 in endothelial cells leads to erythropoietin- and BMP4-independent splenic erythropoiesis in mice.

doi: 10.1371/journal.pone.0055095

Figure Lengend Snippet: Figure 4. Initial splenic erythropoiesis is not mediated by erythropoietin or BMP4. Addition of BMP4 to methylcellulose cultures did not increase the number of colonies compared with erythropoietin alone (A). Levels of BMP4 mRNA (B) and protein (C) were unchanged in the spleen. Splenic BMP4+ macrophages recruited in response to chemotherapeutically-induced anemia (D) were not found in spleens of control (E) or myrAkt1 (F) animals (40x). Plasma erythropoietin protein was not significantly elevated until 4 weeks off tetracycline (E) p,0.0005, and levels of erythropoietin mRNA were not elevated until 3 weeks off tetracycline in the kidney (F) or liver (G) p,0.05. Endothelial cells isolated from myrAkt1 kidneys displayed downregulation of phosphorylated and total Akt1 in the presence of tetracycline (J). TER119+ erythroblasts showed no significant decrease in Annexin V positivity in spleen or bone marrow (K), but TUNEL staining showed a transient decrease in apoptotic nuclei in myrAkt1 spleens at one week p,0.006 (L). doi:10.1371/journal.pone.0055095.g004

Article Snippet: In some cases, the cultures were supplemented with 15 ng/mL mouse BMP4 (R&D Systems).

Techniques: Control, Clinical Proteomics, Isolation, TUNEL Assay, Staining