Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: Basal CALCRL/RAMP associations on RUASM cells. RUASM cells were sparsely grown on 16-well Lab-Tek chamber slides and subjected to PLA. A ) Representative images of PLA for associations of CALCRL with RAMP1, CALCRL with RAMP2, and CALCRL with RAMP3 on RUASM cells incubated with or without primary antibodies against CALCRL, RAMP1, RAMP2, and RAMP3 or without primary antibodies (Neg). B ) Comparison of the red spots per nucleus (blue, 4 ,6-diamidino-2-phenylindole [DAPI]) on RUASM cells are shown in bar graph as mean ± SEM of five random fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Different letters at the top of the bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Incubation, Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: CALCRL/RAMP associations on the RUASM cells with knockdown of RAMP1. Representative images of PLA for associations of CALCRL with RAMP1, CALCRL with RAMP2, and CALCRL with RAMP3 in cells transfected with scramble RAMP1 RNA or shRAMP1 RNA. Red spots per nucleus on cells are presented as bar graphs for mean ± SEM of five random fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Aterisks at the top of the bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Knockdown, Transfection, Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: The effect of CALCB, ADM, and ADM2 on basal CALCRL with RAMP1 associations on RUASM cells. A ) Representative images of PLA for associations of CALCRL with RAMP1 on RUASM cells with or without CALCB, ADM, and ADM2 treatments (100 nM for 4 h). B ) Comparison of the red spots per nucleus for CALCRL with RAMP1 on RUASM cells. Data shown are the means ± SEM of five randomly selected fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. No significant difference was observed between groups ( P > 0.05).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: The effect of CALCB on the associations of CALCRL with RAMP1 on RUASM cells exposed to TNFα. A ) Representative images of PLA for associations of CALCRL with RAMP1 on RUASM cells with or without TNFα (10 ng/ml, 4 h) and CALCB treatments (100 nM for 4 h). Original magnification: ×100 for images in the top panel, ×400 for images in the bottom panel. B ) Comparison of the red spots per nucleus on RUASM cells. Data shown are the means ± SEM of five randomly selected fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Different letters at the top of the bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: The influence of ADM on CALCRL and RAMP1 associations on RUASM cells exposed to TNFα. Representative images of PLA for association of CALCRL with RAMP1 on RUASM cells in controls ( A ), those exposed to TNFα (10 ng/ml) ( B ), or those exposed to TNFα + ADM (100 nM) ( C ) for 4 h. Bar graph shows the comparison of the red spots per nucleus on RUASM cells. Data are the means ± SEM of five randomly selected fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Different letters at the top of the bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: The effect of ADM2 on CALCRL and RAMP1 associations on RUASM cells exposed to TNFα. Representative images of PLA for associations of CALCRL with RAMP1 on RUASM cells in control ( A ), those exposed to TNFα (10 ng/ml) ( B ), or those exposed to TNFα + ADM (100 nM) ( C ) for 4 h. Bar graph shows the comparison of the red spots per nucleus on RUASM cells. Data are the means ± SEM of five randomly selected fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Different letters at the top of the bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Control, Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: Time-dependent rescue effects of CALCB on the associations of CALCRL with RAMP1 on RUASM cells exposed to TNFα for 4 h. The representative images of PLA for associations of CALCRL with RAMP1 on RUASM cells in controls ( A ), those exposed to TNFα (10 ng/ml) for 4 h ( B ), or those exposed to TNFα with addition of CALCB (100 nM) during the last 2 min ( C ), 10 min ( D ), 30 min ( E ), 1 h ( F ), or 4 h ( G ) prior to the termination of incubations at 4 h, respectively. Bar graph shows the comparison of the red spots per nucleus on RUASM cells, presented as mean ± SEM of five random fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Different letters at the top of the bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: Time-dependent effect of coincubation of CALCB with TNFα or TNFα alone for 24 h on the associations of CALCRL with RAMP1 on RUASM cells. RUASM cells treated with TNFα or simultaneously with both TNFα (10 ng/ml) and CALCB (100 nM) for 1, 5, 10, and 30 min, and 1, 2, 4, and 24 h, respectively. Association of CALCRL with RAMP1 on cells measured as red spots per nucleus in different groups are presented as mean ± SEM of five randomly selected fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Different letters at the top of bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Comparison

Journal: Biology of Reproduction

Article Title: Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha 1

doi: 10.1095/biolreprod.116.143529

Figure Lengend Snippet: Effect of LY294002 and PD98059 on the associations of CALCRL with RAMP1 on RUASM cells. Representative images of PLA for associations of CALCRL with RAMP1 on RUASM cells in control ( A ), in the presence of CALCB (100 nM, B ), LY294002 (10 μM, C ), PD98059 (10 μM, D ), TNFα (10ng/ml, E ), TNFα (10 nM) + CALCB (100 nM, F ), TNFα + CALCB + LY294002 ( G ), or TNFα + CALCB + PD98059 ( H ) for 24 h. Bar graph shows comparison of the red spots per nucleus on RUASM cells between groups. Data are the means ± SEM of five random fields from three replicates. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison tests. Different letters at the top of the bars indicate significant differences between groups ( P < 0.01).

Article Snippet: Vascular smooth muscle cells (passages 5–6) from the uterine artery were transfected with a 19-nt short hairpin RNA (shRNA) sequence specific for rat RAMP1 (Origene) by nucleofection, using a nucleofection kit specific for primary smooth muscle cell, per manufacturer's instructions (Lonza).

Techniques: Control, Comparison

Journal: Journal of the American Heart Association

Article Title: miR‐17/20 Controls Prolyl Hydroxylase 2 (PHD2)/Hypoxia‐Inducible Factor 1 (HIF1) to Regulate Pulmonary Artery Smooth Muscle Cell Proliferation

doi: 10.1161/jaha.116.004510

Figure Lengend Snippet: Figure 2. miR-17~92 directly targets PHD2. A, The putative binding site of miR-17 and miR-20a in the 30-UTR of PHD2 and PHD3. B, The expression levels of PHD1, PHD2, and PHD3 proteins were determined in cell lysates of normal hPASMC infected with control lentiviruses (pLVX/Ctrl) or lentiviruses encoding the whole cluster of miR-17~92 (miR-17~92). Tubulin was used as the internal control. C, We determined the protein levels of PHD2 in freshly isolated mPASMC from miR-17~92fl/fland sm- 17~92/ mice. In each strain mPASMC from at least 3 mice were subjected to Western blotting. Representative blots are shown in the top panel and quantification in the bottom panel. D through F, Normal hPASMC were treated with miR-17 and miR- 20a mimics as well as negative control (Neg) and harvested 48 hours after transfection to determine the levels of PHD1 (D), PHD2 (E), and PHD3 (F). The amount of tubulin was used as loading control. n=3. G through I, Normal hPASMC were treated with miR-17 and miR-20a inhibitors or negative control (Neg) and harvested 48 hours after transfection to determine the levels of PHD1 (G), PHD2 (H), and PHD3 (I). The amount of tubulin was used as loading control. n=5. J, Diagram of the wild-type (Wt-Luc) and mutated (Mut-Luc) 30-UTR of PHD2 luciferase reporter constructs. Open triangle represents the binding site of miR-17/20a, and the solid triangle represents the mutated miR-17/20a binding site. K through N, Normal hPASMC were cotransfected with the Wt-Luc reporter (K and L) or Mut-Luc (M and N) and inhibitors (K and M) or mimics (L and N) of miR-17 and miR-20a. n=3 for C through I, 9 for K and L, 5 for M, 4 for N. hPASMC indicates human pulmonary artery smooth muscle cell; mPASMC, mouse pulmonary artery smooth muscle cell; PHD, prolyl hydroxylase. *P<0.05; **P<0.01. Data are presented as meanSEM.

Article Snippet: For overexpression of PHD1 and 2, hPASMC were plated in 100-mm dishes and transfected with 10 lg pEGFP-N1-PHD1 and pEGFP-N1-PHD2 plasmids (Addgene, Cambridge, MA) using an Amaxa Nucleofector II device and an Amaxa Basic Nucleofector Kit for Primary Smooth Muscle Cells (Lonza).

Techniques: Binding Assay, Expressing, Infection, Control, Isolation, Western Blot, Negative Control, Transfection, Luciferase, Construct