Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Inhibition of MicroRNA‐155 Supports Endothelial Tight Junction Integrity Following Oxygen‐Glucose Deprivation

doi: 10.1161/JAHA.118.009244

Figure Lengend Snippet: Efficiency of microRNA‐155 (miR‐155) inhibition and overexpression. A, Diagram describing the experimental setup. Human primary brain microvascular endothelial cells (HBMECs) seeded in cell culture inserts were subjected to 3 hours of oxygen‐glucose deprivation (OGD) and returned back to the normal cell culture conditions; 24 hours later, the cells were transfected with miR‐155 inhibitor, mimic, or appropriate scrambled oligonucleotide. Cells were analyzed at 48 hours after the transfection. B, Fluorescence confocal microscopy of HBMEC s transfected with fluorescein‐labeled miR‐155 inhibitor control (green dots) and stained for actin with rhodamine‐conjugated fluorescent phalloidin. Left panel: orthogonal image projection verifies that fluorescent probes were incorporated within the cell. Bar: 10 μm. C and D, miR‐155 PCR analysis. Total RNA was isolated from the cells subjected to OGD and transfected with the following oligonucleotides: miR‐155 mimic ( OGD /M; grey bar); mimic control ( OGD / MC; grey bar with white stripes); specific miR‐155 inhibitor ( OGD /I; black bar); and control inhibitor ( OGD / IC ; black bar with white stripes). C, P =0.002, (D) P =0.029, Mann–Whitney (Wilcoxon) test. n=3 (for OGD / MC and OGD / IC groups) and n=4 (for OGD /I and OGD /M groups) independent experiments. E, Validation of miR‐155 inhibition by the quantitative assessment of miR‐155 direct target proteins Rictor and SMAD ‐1. Optical density of the protein bands was measured using ImageJ software, normalized to GAPDH density in every sample, and expressed as average relative density values. Protein levels were compared between OGD /I (black bars) and OGD / IC (black bars with white stripes) cell lysates. P =0.029, Mann–Whitney (Wilcoxon) test, n=3 independent experiments per group. Representative immunoblots demonstrate Rictor and SMAD ‐1 protein expression (3 bands per group); GAPDH was used as a loading control. Error bars: SEM ; * P <0.05; ** P <0.01.

Article Snippet: SMAD‐1 and Rictor proteins were detected using SMAD proteins (Cat# 12656) and mTOR pathway (Cat# 9964) antibody sampler kits (Cell Signaling).

Techniques: Inhibition, Over Expression, Cell Culture, Transfection, Fluorescence, Confocal Microscopy, Labeling, Control, Staining, Isolation, MANN-WHITNEY, Biomarker Discovery, Software, Western Blot, Expressing

Journal: Nature Communications

Article Title: R-Spondin 2 governs Xenopus left-right body axis formation by establishing an FGF signaling gradient

doi: 10.1038/s41467-024-44951-7

Figure Lengend Snippet: a Scheme of cell surface competitive binding assays in ( b – e ). b Representative images of HEK293T cells transfected and treated as indicated. Scale bar, 1 mm. c Quantification of ( b ). d Representative images of HEK293T cells transfected and treated as indicated. Scale bar, 1 mm. e Quantification of ( d ). f Amino acid sequence comparison of TK-KC peptides in RSPO2 TSP1 domain of several species. Note that TK-KC peptide sequence is highly conserved (Magenta boxes). g Amino acid sequence comparison of RSPO1-4 TSP1 domains. Note that TK-KC peptide sequence derived from human RSPO2 TSP1 domain is unconserved in other RSPOs (Magenta boxes). h Microinjection strategy for ( i–k ). i Western blot analysis of phosphorylated ERK1/2 and Smad1 (pERK1/2 and pSmad1) and total ERK1/2 and Smad1 (tERK1/2 and tSmad1) in Xenopus embryo (St. 15) lysates. j WISH of dand5 in Xenopus LRO (St. 19) injected as indicated. Arrowheads, dand5 derepression. Scale bar, 100 µm. k Quantification of ( j ). Two-sided Fisher’s exact test used for statistical analysis. n = number of dorso-posterior explants. l Model showing the mode of action for TK to intervene RSPO2-FGFR4 interaction and increases FGFR4 signaling. Data information: For all cell surface binding assays ( c , e ), data are displayed as mean ± SD with two-tailed unpaired t -test: n = 3 biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used for western blotting: Rabbit anti-Phospho-ERK1/2 (Cell Signaling Technology 9101 S); Rabbit anti-ERK1/2 (Sigma M5670); Mouse anti-beta-Catenin (BD 610154); Rabbit anti-LRP6 (Cell Signaling Technology 2560); Rabbit anti-GAPDH (Cell Signaling Technology 2118 S); Goat anti-RSPO2 (R and D systems AF3266); Rat anti-HA (Roche 11867423001); Rabbit anti-Phospho-Smad1 (Cell Signaling Technology 9516); Rabbit anti-Smad1(Cell Signaling Technology 9743 S).

Techniques: Binding Assay, Transfection, Sequencing, Comparison, Derivative Assay, Western Blot, Injection, Two Tailed Test

Journal: Oncotarget

Article Title: Oncogenic features of the bone morphogenic protein 7 (BMP7) in pheochromocytoma

doi:

Figure Lengend Snippet: A. IHC or IF on adrenal medullary tissue from wild-type and mutant rats was performed using a rat anti-BMP7 antibody or an anti-P-Smad1/5/8 antibody, respectively. These tissues are representative of five tissues per animal group. Original magnification: 400× B. Plasma Bmp7 levels were measured in seven MENX mutant rats and seven wild-type rats fasted for 12 h. Measurements were performed using a rat Bmp7 ELISA-KIT. ***, P < 0.001.

Article Snippet: The primary antibodies used are: human anti-BMP7 (R&D Systems, USA; clone #164311; dilution 1:1000); rat anti-Bmp7 (#4693, 1:100), P-Smad1/5/8 (#9511, 1:50), Smad1 (#5753; 1:500), AKT (#9272; 1:500) and P-AKT (#9271; 1:500) all from Cell Signaling Technology (Danvers MA, USA); integrin β1 (Abcam, Cambridge, UK; #EP1041Y; dilution 1:500); anti-Myc tag (Clontech, St-Germain-en-Laye, France; #631206; dilution 1:500), α-Tubulin (Sigma-Aldrich, Hamburg, Germany; #T5168; dilution 1:1000).

Techniques: Mutagenesis, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Oncogenic features of the bone morphogenic protein 7 (BMP7) in pheochromocytoma

doi:

Figure Lengend Snippet: A. PC12 cells were transfected with a BMP7-containing (BMP7) or an empty (mock) vector. Twenty-four h post transfection IF was performed using specific antibodies targeting integrin β1 (1:400) or BMP7 (1:100). Cell nuclei were counterstained with DAPI. In parallel, the expression of integrin β1 and BMP7 proteins was determined by western blotting using specific antibodies. α-Tubulin was used as loading control. B. PC12 cells were either transfected (txBMP7) with a Myc-BMP7 plasmid or treated with 100 ng/mL recombinant human BMP7 (rhBMP7). After 24 h, cells were collected and analyzed by western blotting using antibodies against P-Smad1/5/8 and Smad1. α-Tubulin was used as a loading control. C. PC12 cells were treated with rhBMP7 for 5, 15, 30, 45, or 60 min. Western blot analysis was performed using specific antibodies raised against integrin β1, AKT and P-AKT. α-Tubulin was used as loading control. D. We co-transfected PC12 cells with BMP7 and scr-siRNA or siRNA- Itgb1 and 24 h later proliferation, migration and invasion were assessed. Values for cells with knockdown of Itgb1 were normalized against the values of scr-siRNA-transfected cells arbitrarily set to 100%. The experiments were performed two times with three technical replicates with similar results. For proliferation, the average of the 2 experiments is shown. For migration/invasion, five random fields of each test at × 400 magnification were counted (average ± SD). **, P < 0.01; ***, P < 0.001. In parallel, the levels of BMP7, integrin β1, P-AKT and AKT were assessed in the transfected cells by western blotting with specific antibodies as described above. α-Tubulin was used as a loading control. E. PC12 cells were treated with the indicated concentrations of the P-AKT inhibitor GSK690693 and western blot analysis was performed 24 h later to determine the expression of BMP7, integrin β1, P-AKT, and AKT. α-Tubulin was used as a loading control. F. Expression of BMP7, integrin β1, and P-AKT in 10 human primary PCCs and one human normal medulla (cont.) was assessed by western blotting using specific antibodies. α-Tubulin was used as a loading control.

Article Snippet: The primary antibodies used are: human anti-BMP7 (R&D Systems, USA; clone #164311; dilution 1:1000); rat anti-Bmp7 (#4693, 1:100), P-Smad1/5/8 (#9511, 1:50), Smad1 (#5753; 1:500), AKT (#9272; 1:500) and P-AKT (#9271; 1:500) all from Cell Signaling Technology (Danvers MA, USA); integrin β1 (Abcam, Cambridge, UK; #EP1041Y; dilution 1:500); anti-Myc tag (Clontech, St-Germain-en-Laye, France; #631206; dilution 1:500), α-Tubulin (Sigma-Aldrich, Hamburg, Germany; #T5168; dilution 1:1000).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Recombinant, Migration, Knockdown

Journal: Oncotarget

Article Title: Oncogenic features of the bone morphogenic protein 7 (BMP7) in pheochromocytoma

doi:

Figure Lengend Snippet: A. MTT cells were treated with DMH1 (3 μM or 5 μM) or with DMSO vehicle. Proliferation was assessed at the indicated times by the WST-1 assay. The experiments were performed two times with six technical replicates each, and are expressed as the mean ± SD. The values are normalized against those of DMSO-treated cells set to 100%. ***, P < 0.001. B. MTT cells treated as in A for 24 h were used for migration assays as indicated in Figure . The percentage of cells that migrated was normalized against the values of DMSO-treated cells arbitrarily set to 100%. The experiment was performed two times with three technical replicates with similar results. Five random fields of each test at ×400 magnification were counted (±SD). *, P < 0.05; **, P < 0.01. C. MTT cells were treated with DMH1 (3 μM or 5 μM) or with DMSO vehicle for 24 h. Proteins were then collected and probed for the expression of integrin β1, P-Smad1/5/8, Smad1, P-AKT and AKT. α-Tubulin was used as a loading control. D. Rat primary tumor cells were treated with DMH1 (3 μM and 5 μM) or DMSO for 72 h and cell viability was assessed using the WST-1 assay. Shown is the average of five independent cultures from five mutant rats (8 months of age). The values are normalized against those of DMSO-treated cells set to 100%. Data were analyzed independently with six technical replicates each and are expressed as the mean ± SD. ***, P < 0.001.

Article Snippet: The primary antibodies used are: human anti-BMP7 (R&D Systems, USA; clone #164311; dilution 1:1000); rat anti-Bmp7 (#4693, 1:100), P-Smad1/5/8 (#9511, 1:50), Smad1 (#5753; 1:500), AKT (#9272; 1:500) and P-AKT (#9271; 1:500) all from Cell Signaling Technology (Danvers MA, USA); integrin β1 (Abcam, Cambridge, UK; #EP1041Y; dilution 1:500); anti-Myc tag (Clontech, St-Germain-en-Laye, France; #631206; dilution 1:500), α-Tubulin (Sigma-Aldrich, Hamburg, Germany; #T5168; dilution 1:1000).

Techniques: WST-1 Assay, Migration, Expressing, Control, Mutagenesis