Journal: Physiological Reports

Article Title: The effect of mitoTEMPO on the development of hypoxia‐induced pulmonary hypertension in male mice

doi: 10.14814/phy2.70804

Figure Lengend Snippet: Effect of mitoTEMPO treatment on HIF‐1α stabilization in vitro. HIF‐1α protein levels were assessed by western blot, and steady‐state mRNA levels for lactate dehydrogenase A ( Ldha ) and pyruvate dehydrogenase kinase 1 ( Pdk1 ) by quantitative real‐time PCR in (a) CMT167 cells, (b) hPASMCs, and (c) mPASMCs, exposed to normoxia (21% O 2 ), severe hypoxia (1% O 2 ), or mild hypoxia (10% O 2 ) for 24 h and treated with triphenylphosphonium (TPP + ) (blue dots) or mitoTEMPO (MT) (red dots). Immunoblots shown are representative of three independent experiments. CMT167: mouse lung carcinoma epithelial cells; hPASMCs: human pulmonary artery smooth muscle cells; mPASMCs: mouse pulmonary artery smooth muscle cells. Densitometric analysis of HIF‐1α bands normalized to β‐Actin. Data are presented as mean ± SD. Statistical comparisons were made using two‐way ANOVA with Tukey's post hoc test ( n = 3 per group). (ns: no signal). Quantitative real‐time PCR data reflect mean ΔCt ± SD ( n = 3 per experimental group).

Article Snippet: Mouse lung carcinoma epithelial (CMT167) cells (10032302, Merck, Germany) and human PASMCs (hPASMCs) (C‐12521, PromoCell, Germany) were purchased.

Techniques: In Vitro, Western Blot, Real-time Polymerase Chain Reaction

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