Journal: Scientific Reports

Article Title: MicroRNA-378a-3p is overexpressed in psoriasis and modulates cell cycle arrest in keratinocytes via targeting BMP2 gene

doi: 10.1038/s41598-021-93616-8

Figure Lengend Snippet: Validation of candidate gene targets of miR-378a. ( a ) The expression levels of BMP2 and INHBA by qPCR using mRNA from NHEK cells transfected with miR-378a mimic or inhibitor for 24 h and 48 h. ( b ) Comparison of the expression levels of the selected candidate genes from high-throughput RNA-sequencing (light colors) versus real-time qPCR (dark colors) from NHEK cells over-expressing miR-378a mimic (red) or inhibitor (blue) for 24 h and 48 h. Error bars indicate mean ± SEM from three replicates. The experiment was performed three times independently. *Indicates P < 0.05, **indicates P < 0.01, and ***indicates P < 0.001.

Article Snippet: The membranes were cut according to molecular weight of the proteins and initially blocked with blocking buffer (5% non-fat dry milk in PBST) for 1 h. Later, it was immunoblotted with anti-BMP2 antibody (1:1000 dilution; #AHP2442, Bio-Rad Laboratories, Inc.), anti-involucrin antibody (1:1000 dilution; #sc-398952, Santa Cruz Biotechnology), and anti-GAPDH (1:5000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA; cat#sc-25778) for 1 h at room temperature.

Techniques: Biomarker Discovery, Expressing, Transfection, Comparison, High Throughput Screening Assay, RNA Sequencing

Journal: Scientific Reports

Article Title: MicroRNA-378a-3p is overexpressed in psoriasis and modulates cell cycle arrest in keratinocytes via targeting BMP2 gene

doi: 10.1038/s41598-021-93616-8

Figure Lengend Snippet: BMP2 as a novel target gene of miR-378a in keratinocytes. ( a ) Hypothetical binding structure of miR- 378a at 3′UTR of BMP2 (left) and INHBA (right) with predictive binding energy and probability value for actual binding. ( b ) Luciferase activity of wild-type and mutant 3′UTR of BMP2 genes in HEK293T cells co-transfected with miR-378a mimic or inhibitor for 48 h. ( c ) Western blot images (top) and quantitative measurement (bottom) of BMP2 protein in NHEK cells with miR-378a overexpression or suppression for 72 h. A representative image of BMP2 protein from four independent experiments was shown (also see Supplementary Fig. ). ( d ) The expression level of BMP2 in psoriatic lesions (n = 4) and healthy individuals (n = 4). *Indicates P < 0.05, **indicates P < 0.01, and ***indicates P < 0.001.

Article Snippet: The membranes were cut according to molecular weight of the proteins and initially blocked with blocking buffer (5% non-fat dry milk in PBST) for 1 h. Later, it was immunoblotted with anti-BMP2 antibody (1:1000 dilution; #AHP2442, Bio-Rad Laboratories, Inc.), anti-involucrin antibody (1:1000 dilution; #sc-398952, Santa Cruz Biotechnology), and anti-GAPDH (1:5000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA; cat#sc-25778) for 1 h at room temperature.

Techniques: Binding Assay, Luciferase, Activity Assay, Mutagenesis, Transfection, Western Blot, Over Expression, Expressing

Journal: Molecular Medicine Reports

Article Title: Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

doi: 10.3892/mmr.2015.4627

Figure Lengend Snippet: Effect of ZA on the expression of BMP-2 and phosphorylation of the ERK 1/2 and p38 pathways in MC3T3-E1 cells. The MC3T3-E1 cells were incubated with various concentrations of ZA (0–1 µ M) for 7 days. (A) Levels of secreted BMP-2, measured using an ELISA. (B) mRNA expression levels of BMP-2, determined using reverse transcription-quantitative polymerase chain reaction analysis. (C) Protein levels of BMP-2, p38, p-p38, ERK 1/2 and p-ERK 1/2, detected using western blot analysis and normalized to GAPDH. (D) Quantitative analysis of the blots for BMP-2. (E) Relative expression of p-ERK, normalized to ERK, and relative expression of p-p38 normalized to p38. The results are expressed as the mean ± standard error of the mean (n =3 for each group). * P<0.05, ** P<0.01 and *** P<0.001, compared with the 0 µ M group. BMP-2, bone morphogenetic protein 2; ERK 1/2, extracellular signal-regulated kinase 1/2; p-, phosphorylated; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Primary antibodies against the following targets were used: Monoclonal rabbit anti-p38 antibody (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 8690), monoclonal rabbit anti-phosphorylated (p)-p38 antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4511), monoclonal rabbit anti-ERK 1/2 antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4695), monoclonal rabbit anti-p ERK 1/2 antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4370), polyclonal rabbit anti-inactive caspase-3 antibody (1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. SC-7148), polyclonal rabbit anti-OCN antibody (1:500; Santa Cruz Biotechnology, Inc.; cat. no. SC-30045), polyclonal rabbit anti-active caspase-3 antibody (1:200; Abcam, Cambridge, UK; cat. no. ab2302), monoclonal rabbit anti-ALP antibody (1:20,000; Abcam; cat. no. ab108337), polyclonal rabbit anti-BMP-2 antibody (1:1,000; Abgent Biotech Co., Ltd., Suzhou, China; cat. no. AP13858c), polyclonal rabbit anti-Runx2 antibody (1:400; Wuhan Boster Biological Technology, Ltd.; cat. no. BA3613-2), monoclonal mouse anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody (1:400; Wuhan Boster Biological Technology, Ltd.; cat. no. BM1623), monoclonal mouse anti-β actin antibody (1:400; Wuhan Boster Biological Technology, Ltd.; cat. no. BM0627).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: RGMa Participates in the Blood–Brain Barrier Dysfunction Through BMP/BMPR/YAP Signaling in Multiple Sclerosis

doi: 10.3389/fimmu.2022.861486

Figure Lengend Snippet: BMP2 and BMPR II expression in EAE mice. (A–F) BMP2 and BMPR II in the brain and spinal cord were both upregulated in the 14- and 21-day group compared with the control group. BMP2 and BMPR II in both the brain and spinal cord were significantly higher in the 21-day than the 0-day groups ( n = 4, error bar: SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Bonferroni). (G–J) Immunofluorescence showed BMP2 and BMPR II were located in CD31 + endothelial cells in both the brain cortex and the white matter of the spinal cord. Scale bar: 50 µm (brain), 25 µm (spinal cord).

Article Snippet: The membranes were then incubated at 4°C overnight with primary antibodies for 16 h. The primary antibodies were as follows: anti-RGMa (1:10,000, ab169761, Abcam, USA); anti-BMP2 (1:1,000, YT5651, Immunoway, USA); anti-BMPR II (1:500, 220550, ZEN Bio, Chengdu); anti-YAP (1:1,000, 14074, Cell Signaling Technology, USA); anti-ZO-1(1:1,000, 40-2200, ThermoFisher Scientific, USA); anti-GAPDH (1:10,000, 20494-1-AP, Proteintech, Wuhan); and anti-beta Tubulin (1:4,000; 10068-1-AP, Proteintech, Wuhan).

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: RGMa Participates in the Blood–Brain Barrier Dysfunction Through BMP/BMPR/YAP Signaling in Multiple Sclerosis

doi: 10.3389/fimmu.2022.861486

Figure Lengend Snippet: BMPR II mediates the integrity of the BBB induced by RGMa. (A, B) Immunofluorescence showed BMP2 and BMPR II were expressed in HBMECs. Scale bar: 50 µm. (C–E) Both BMP2 and BMPR II expression levels were significantly increased after overexpressing RGMa ( n = 3, * p < 0.05, t -test). (F–I) On the basis of overexpressing RGMa, specifically silencing BMPR II significantly augments ZO-1 and claudin-5 expression ( n = 3, * p < 0.05, t -test). **p < 0.01.

Article Snippet: The membranes were then incubated at 4°C overnight with primary antibodies for 16 h. The primary antibodies were as follows: anti-RGMa (1:10,000, ab169761, Abcam, USA); anti-BMP2 (1:1,000, YT5651, Immunoway, USA); anti-BMPR II (1:500, 220550, ZEN Bio, Chengdu); anti-YAP (1:1,000, 14074, Cell Signaling Technology, USA); anti-ZO-1(1:1,000, 40-2200, ThermoFisher Scientific, USA); anti-GAPDH (1:10,000, 20494-1-AP, Proteintech, Wuhan); and anti-beta Tubulin (1:4,000; 10068-1-AP, Proteintech, Wuhan).

Techniques: Immunofluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: RGMa Participates in the Blood–Brain Barrier Dysfunction Through BMP/BMPR/YAP Signaling in Multiple Sclerosis

doi: 10.3389/fimmu.2022.861486

Figure Lengend Snippet: RGMa regulates BMP2 and thereby tight junctional protein expression. (A–C) BMP2 and BMPR II expression were significantly decreased after RGMa knockdown in vitro BBB ( n = 3, * p < 0.05, t -test). (D–G) After activating BMPR II by mnTBAP 100 µM for 1 h based on inhibiting RGMa, ZO-1 and claudin-5 expression levels were significantly reduced ( n = 3, * p < 0.05, t -test). ****p < 0.0001.

Article Snippet: The membranes were then incubated at 4°C overnight with primary antibodies for 16 h. The primary antibodies were as follows: anti-RGMa (1:10,000, ab169761, Abcam, USA); anti-BMP2 (1:1,000, YT5651, Immunoway, USA); anti-BMPR II (1:500, 220550, ZEN Bio, Chengdu); anti-YAP (1:1,000, 14074, Cell Signaling Technology, USA); anti-ZO-1(1:1,000, 40-2200, ThermoFisher Scientific, USA); anti-GAPDH (1:10,000, 20494-1-AP, Proteintech, Wuhan); and anti-beta Tubulin (1:4,000; 10068-1-AP, Proteintech, Wuhan).

Techniques: Expressing, In Vitro

Journal: Frontiers in Immunology

Article Title: RGMa Participates in the Blood–Brain Barrier Dysfunction Through BMP/BMPR/YAP Signaling in Multiple Sclerosis

doi: 10.3389/fimmu.2022.861486

Figure Lengend Snippet: A proposed model of RGMa regulation of BMP2/BMPR II/YAP signaling in endothelial cells. The overexpression of RGMa forms a complex by binding BMP2 and BMPR II, then attenuates YAP expression, which promotes the disruption of the BBB. By regulating the BMP2/BMPR II/YAP pathway, RGMa mediates dysfunction of the blood–brain barrier.

Article Snippet: The membranes were then incubated at 4°C overnight with primary antibodies for 16 h. The primary antibodies were as follows: anti-RGMa (1:10,000, ab169761, Abcam, USA); anti-BMP2 (1:1,000, YT5651, Immunoway, USA); anti-BMPR II (1:500, 220550, ZEN Bio, Chengdu); anti-YAP (1:1,000, 14074, Cell Signaling Technology, USA); anti-ZO-1(1:1,000, 40-2200, ThermoFisher Scientific, USA); anti-GAPDH (1:10,000, 20494-1-AP, Proteintech, Wuhan); and anti-beta Tubulin (1:4,000; 10068-1-AP, Proteintech, Wuhan).

Techniques: Over Expression, Binding Assay, Expressing