Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 suppress HPASMC proliferation and migration. HPASMCs were cultured with specific microRNAs (miRNAs) as indicated for 48 h under hypoxic culture conditions, followed by qRT‒PCR. ( B , C ) Cells from ( A ) were seeded into 96-well plates, followed by MTT ( B ) and EdU ( C ), the statistical analysis were performed. D Cells from ( A ) were subjected to a transwell assay, the statistical analysis were performed.The data are presented as the means ± SDs ( n = 5). The statistical test used was the paired t test. ***, p < 0.001 versus miR-NC.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Cell Culture, Transwell Assay

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 inhibit the expression of TRIM29. ( A ) HPASMCs were transfected with miR-637 or miR-661, followed by RNA-seq analysis. Venn diagram of gene changes after the overexpression of two different microRNAs (FC: fold change). ( B , C ) HPASMCs were overexpressing miR-637 or miR-661, followed by RNA-seq analysis. The transcript volcano plot shows the differentially expressed genes as indicated. ( D ) HPASMC cells were transfected with indicated plasmids for 48 h, followed by luciferase assay. ( E ) HPASMCs were overexpressing miR-637 or miR-661 under hypoxic culture conditions, followed by RT‒PCR and WB. Original blots are presented in Supplementary Fig. 1.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Transfection, RNA Sequencing, Over Expression, Luciferase

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: TRIM29 attenuates the miR-637- and miR-661-induced inhibition of AKT/mTOR signalling. ( A ) Statistical analysis of the results of the GO enrichment assay was performed after the indicated miRNAs were overexpressed. B-C HPASMCs were cultured for different durations under hypoxic conditions, followed by WB ( B ) and qRT‒PCR ( C ). ( D ) qRT‒PCR was used to detect the expression level of TRIM29 in the serum of patients with pulmonary hypertension and healthy volunteers. ( E ) HPASMCs were overexpressing miR-637 or miR-661 under hypoxic culture conditions, followed by RT‒PCR and WB. ( F ) WB assays were performed after the inhibition of miR-637 and miR-661. ( G ) HPASMCs were transfected with Flag-TRIM29 for 48 h under hypoxic culture conditions, and the cells were subjected to WB analysis. ( H ) TRIM29 was knocked out in HAPSMCs under hypoxic culture conditions, and cell lysates were then prepared for WB analysis. ( I ) HPASMCs were transfected with Flag-TRIM29 or overexpressed with microRNA as indicated for 48 h under hypoxic culture conditions, followed by WB analysis. Original blots are presented in Supplementary Figs. 2–7.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Cell Culture, Expressing, Transfection

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 attenuate TRIM29-induced HPASMC proliferation and migration. ( A ) HPASMCs were transfected with the indicated plasmids or infected with TRIM29 sgRNA for 48 h of hypoxia, followed by the MTT assay. ( B – E ) HPASMCs were transfected with Flag-TRIM29, infected with TRIM29 sgRNA lentivirus or overexpressed with microRNA as indicated for 48 h under hypoxic culture conditions, followed by the EdU assay ( B ), wound healing assay ( D ) and the statistical analysis were performed ( C , E ). The data are presented as the means ± SDs ( n = 5). The statistical test used was the paired t test. ***, p < 0.001 versus miR-NC.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Transfection, Infection, MTT Assay, EdU Assay, Wound Healing Assay

Journal: Cells

Article Title: Stress Granule Assembly in Pulmonary Arterial Hypertension

doi: 10.3390/cells13211796

Figure Lengend Snippet: SG puncta in pulmonary blood vessels in lungs from patients with PAH compared to controls; PASMCs from the same patients have more SGs after acute oxidative stress and G3BP1 downregulation decreases their proliferation compared to controls. ( A ) Paraffin-embedded sections of lungs from patients with PAH and healthy donors stained with G3BP1. Arrows indicate G3BP1 puncta, quantified with CellProfiler ( B ). ( C ) PASMCs from patients with PAH and healthy donors were treated with 0.5 mM arsenite for 40 min and stained for G3BP1 to detect SGs, quantified with CellProfiler ( D ). Nuclei were visualized with DAPI staining. Scale bar, 20 μm; **** p < 0.0001 ( n = 45 cells/cell type). ( E ) Immunoblots confirming downregulation of G3BP1 in hPASMCs isolated from patients with PAH and healthy donors. Knockdown of G3BP1 inhibits synthetic marker (Connexin 43) and increases contractile marker (desmin) expression in PASMCs isolated from patients with PAH and healthy donors. ( F ) Knockdown of G3BP1 decreased the number of HPASMCs isolated from patients with PAH compared to healthy donors at 72 h (Cell proliferation reagent WST-1); ** p < 0.01, **** p < 0.0001.

Article Snippet: Human Pulmonary Arterial Smooth Muscle Cells (HPASMCs) were cultured in complete SmBM Lonza media supplemented with antibiotic/antimycotic solution 1:100 and Rat Pulmonary Arterial Smooth Muscle Cells (RPASMCs) were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin and were grown at 37 °C in 95% humidified air with 5% CO 2 to 70% confluency prior to experimentation.

Techniques: Staining, Western Blot, Isolation, Knockdown, Marker, Expressing