Journal: International Journal of Molecular Sciences

Article Title: ACVR1, a Therapeutic Target of Fibrodysplasia Ossificans Progressiva, Is Negatively Regulated by miR-148a

doi: 10.3390/ijms13022063

Figure Lengend Snippet: Interaction of miR-148a with the 3′ UTR of ACVR1 mRNA. ( A ) Schema of human ACVR1 mRNA and miR-148a binding site in its 3′-UTR sequence. ( B ) Schematic indication of miR-148a binding sites in the 3′ UTR of ACVR1 mRNA based on TargetScan 5.1 prediction (seed sequences at 448–454 bp of ACVR1 3′ UTR were highlighted in grey, mutant bases for vector pmir-ACVR1-3′UTR-mut construction were shown as lowercase letters). ( C ) Multiple sequence alignment of the miR-148a binding sites in different species. The alignment was performed with ClustalW. The miR-148a seed sequences at 448–454 bp of ACVR1 3′ UTR were framed. * conservative site. ( D ) Luciferase reporter vector structure. The vector contained two expression units; one for the firefly luciferase gene (luc2) and the other for the Renilla luciferase gene (hRluc-neo fusion) expression. Luc2 unit was driven by a human phosphoglycerate kinase (PGK) promoter and contained multiple cloning sites (MCS) downstream of the luc2 sequence. ACVR1 3′ UTR containing a putative miRNA target region was cloned into the MCS. The Renilla luciferase unit was driven by an SV40 early promoter and used to adjust for variations in transfection efficiency among experiments and normalize firefly luciferase activity.

Article Snippet: Antibodies directed against ACVR1(sc-25449) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA); goat anti-mouse(GAM)-HRP conjugate (# 170-5047) and goat anti-rabbit (GAR)-HRP conjugate (# 170-5046) were purchased from Bio-Rad. β-actin was used as a loading control.

Techniques: Binding Assay, Sequencing, Mutagenesis, Plasmid Preparation, Luciferase, Expressing, Clone Assay, Transfection, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: ACVR1, a Therapeutic Target of Fibrodysplasia Ossificans Progressiva, Is Negatively Regulated by miR-148a

doi: 10.3390/ijms13022063

Figure Lengend Snippet: miR-148a directly targets human ACVR1 3′ UTR. Relative luciferase activity derived from pmirGLO-ACVR1-3′UTR or pmirGLO-ACVR1-3′UTR-mut were co-transfected with miR-148a mimic or NC mimic in HeLa cells. The transfection of pmirGLO-ACVR1-3′UTR or pmirGLO-ACVR1-3′UTR-mut alone was used as control (Mock). Firefly and Renilla luciferase activities were determined, and firefly luciferase was normalized to Renilla luciferase activity. Results are expressed as relative activities against the activity of mock mimic transfection. ** p < 0.01.

Article Snippet: Antibodies directed against ACVR1(sc-25449) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA); goat anti-mouse(GAM)-HRP conjugate (# 170-5047) and goat anti-rabbit (GAR)-HRP conjugate (# 170-5046) were purchased from Bio-Rad. β-actin was used as a loading control.

Techniques: Luciferase, Activity Assay, Derivative Assay, Transfection

Journal: International Journal of Molecular Sciences

Article Title: ACVR1, a Therapeutic Target of Fibrodysplasia Ossificans Progressiva, Is Negatively Regulated by miR-148a

doi: 10.3390/ijms13022063

Figure Lengend Snippet: Verification of target genes of miR-148a. ( A ) QRT-PCR results of miR-148a expression level in HeLa cells transfected with miR-148a mimic or NC mimic for 48 h. RNU44 (RNA, U44 small nuclear) was used as the normalization control. ( B ) QRT-PCR results of mRNA level of ACVR1 in HeLa cells transfected as described in A. GAPDH was used as the normalization control. ( C ) Western blot analysis of ACVR1 protein level in HeLa cells transfected as described in A for 72 h. ( D ) The bands’ intensity in C is quantified using the ImageJ 1.43 software . The relative intensity against β-actin was calculated, and fold change relative to the relative intensity in transfected NC cells is presented.

Article Snippet: Antibodies directed against ACVR1(sc-25449) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA); goat anti-mouse(GAM)-HRP conjugate (# 170-5047) and goat anti-rabbit (GAR)-HRP conjugate (# 170-5046) were purchased from Bio-Rad. β-actin was used as a loading control.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Software