Journal: Respiratory Research

Article Title: TRB3 mediates vascular remodeling by activating the MAPK signaling pathway in hypoxic pulmonary hypertension

doi: 10.1186/s12931-021-01908-4

Figure Lengend Snippet: Effect of TRB3 knockdown on proliferation, migration, and apoptosis of hypoxic PASMCs. PASMCs were transfected with lentiviral vectors encoding short hairpin RNAs targeting against rat TRB3(si-TRB3) or negative control(si-NC) for further analysis. A CCK-8 assays were performed to test cell viability after TRB3 knockdown in hypoxic PASMCs. B The EdU proliferation assay was used to measure cell proliferation. All images are × 400 magnification. C EdU‐positive cells were analyzed. D and E The expression of PCNA and Survivin was examined by western blot, and the gray value was quantified by ImageJ software. F Cell apoptosis was evaluated by Hoechst 33342 staining, and the percentage of apoptosis was quantified. G Crystal violet staining of PASMCs is presented at × 200 magnification for the Transwell assay, and the migrated cells were counted manually. H Apoptosis-related protein expression was evaluated and quantification of the analysis is shown. I The expression of MMP9 was evaluated. Data represent the mean ± SEM (n = 4). *P < 0.05 and **P < 0.01 compared to normoxic PASMCs transfected with si-NC. # P < 0.05 and ## P < 0.01 compared to hypoxic PASMCs transfected with si-TRB3. si-NC, lentiviral vectors encoding the negative control sequence; si-TRB3, lentiviral vectors encoding the short-hairpin RNAs against rat TRB3; PCNA, proliferating cell nuclear antigen; TRB3, Tribbles homolog 3; BAX, BCL2 associated X; Bcl2, B cell leukemia/lymphoma 2; MMP9, matrix metallopeptidase 9

Article Snippet: Paraffin sections of the lung were stained with hematoxylin–eosin (HE), anti-α-smooth muscle actin (α-SMA) (1:100; Boster Biological Technology, Wuhan, China), anti-proliferating cell nuclear antigen (PCNA) (1:100; Proteintech Group), and anti-TRB3 (1:100; Proteintech Group).

Techniques: Knockdown, Migration, Transfection, Negative Control, CCK-8 Assay, Proliferation Assay, Expressing, Western Blot, Software, Staining, Transwell Assay, Sequencing

Journal: Respiratory Research

Article Title: TRB3 mediates vascular remodeling by activating the MAPK signaling pathway in hypoxic pulmonary hypertension

doi: 10.1186/s12931-021-01908-4

Figure Lengend Snippet: Inhibitors of ERK, JNK, and p38 MAPK reversed the effect of TRB3 overexpression on PASMC biological behavior. A The CCK-8 assay was used to evaluated the proliferation of TRB3-overexpressing cells after treating with U0126, SP600125, and SB203580. B – D Western blot analysis of PCNA expression in TRB3 overexpressing cells incubated with U0126, SP600125, or SB203580 for 12 h. E Cell apoptosis induced by U0126, SP600125 and SB203580 in TRB3-overexpressing cells was evaluated using flow cytometry, and the percentage of early apoptotic (Annexin V+/PI−) and late apoptotic (Annexin V+ /PI+) cells was analyzed. F – H Western blot analysis of the protein expression of PARP, BAX, and Bcl2 in TRB3-overexpressing cells incubated with U0126, SP600125, and SB203580 for 12 h. I Crystal violet staining is presented at × 200 magnification for the Transwell assay and the migrated cells were counted and analyzed. J – L Western blot analysis of MMP9 expression in TRB3-overexpressing cells incubated with U0126, SP600125, and SB203580 for 12 h. Data represent the mean ± SEM (n = 4). *P < 0.05 compared to normoxic PASMCs transfected with lv-NC. # P < 0.05 and ## P < 0.01 compared to PASMCs transfected with lv-TRB3. U0126, an ERK signaling inhibitor; SP600125, an JNK signaling inhibitor; SB203580, an p38 MAPK signaling inhibitor

Article Snippet: Paraffin sections of the lung were stained with hematoxylin–eosin (HE), anti-α-smooth muscle actin (α-SMA) (1:100; Boster Biological Technology, Wuhan, China), anti-proliferating cell nuclear antigen (PCNA) (1:100; Proteintech Group), and anti-TRB3 (1:100; Proteintech Group).

Techniques: Over Expression, CCK-8 Assay, Western Blot, Expressing, Incubation, Flow Cytometry, Staining, Transwell Assay, Transfection

Journal: Respiratory Research

Article Title: TRB3 mediates vascular remodeling by activating the MAPK signaling pathway in hypoxic pulmonary hypertension

doi: 10.1186/s12931-021-01908-4

Figure Lengend Snippet: TRB3 knockdown reversed and prevented the progression of hypoxia -induced PH. A The right ventricular systolic pressure(RVSP) was measured by a 1.2-Fr pressure catheter (GuGeShengWu, Wuhan, China) inserted via the right jugular vein and positioned in the right ventricle. B The values of RVSP were obtained using the ADInstruments Powerlab data acquisition system. C Right ventricular hypertrophy was evaluated by the ratio of the RV/(LV + S). The right ventricle (RV) was peeled off from the left ventricle (LV) and the septum (S), and the dry weight of the two components was measured. D Systemic blood pressure was measured by a 1.2-Fr pressure catheter inserted via the left carotid artery. E Lung tissue sections were stained for hematoxylin–eosin (HE) (magnification, × 200) and α-SMA (magnification, × 400). F Pulmonary vascular remodeling was evaluated by the percentage of medial wall thickness of distal pulmonary vessels, which was expressed using the following equation: [(external diameter − internal diameter)/external diameter]. G The whole pulmonary artery of each rat from different groups was extracted, and TRB3 protein expression in each group was measured by western blot analysis. H and I PCNA protein expression in pulmonary arteries was examined by immunohistochemical staining and western blot analysis. J MMP9 protein expression in pulmonary arteries was examined by western blot analysis. K The phosphorylated forms of ERK, JNK, and p38 MAPK in pulmonary arteries in each group were evaluated by western blot analysis. Data represent the mean ± SEM (n = 4). *P < 0.05 and **P < 0.01

Article Snippet: Paraffin sections of the lung were stained with hematoxylin–eosin (HE), anti-α-smooth muscle actin (α-SMA) (1:100; Boster Biological Technology, Wuhan, China), anti-proliferating cell nuclear antigen (PCNA) (1:100; Proteintech Group), and anti-TRB3 (1:100; Proteintech Group).

Techniques: Knockdown, Staining, Expressing, Western Blot, Immunohistochemical staining

Journal: Pulmonary Circulation

Article Title: Transmembrane protein 16A/anoctamin 1 inhibitor T16A inh -A01 reversed monocrotaline-induced rat pulmonary arterial hypertension

doi: 10.1177/2045894020946670

Figure Lengend Snippet: Upregulation of TMEM16A expression in the PAs of PAH-modeling rats and its correlation with PCNA and WA%. Representative Western blots are shown. Pulmonary arterial tissues were obtained for Western blots from rats treated with MCT for 0 (control), 1 (1W), 2 (2W), 3 (3W), and 4 weeks (4W). Representative Western blot band of TMEM16A (a), PCNA (b), P-ERK1/2 (c) of pulmonary arterial tissues from rats treated with MCT for 0 (control), 1 (1W), 2 (2W), 3 (3W), and 4 weeks (4W). (d) to (f) Analytic data of TMEM16A, PCNA, P-ERK1/2 protein expression. (g) TMEM16A mRNA expression of pulmonary arterial tissues from rats treated with MCT for 0 (control), 1 (1W), 2 (2W), 3 (3W), and 4 weeks (4W). **: P < 0.01 versus control. n = 6 for each group. ERK1/2: extracellular regulated protein kinases; NS: not significant; PCNA: proliferating cell nuclear antigen; TMEM16A: transmembrane protein 16A.

Article Snippet: The proteins were separated on the gel and then transferred onto polyvinylidene difluoride membranes, which were blocked with 5% skimmed milk for 2 h and immunoblotted with a primary antibody against TMEM16A (Abcam, Cambridge, UK) at a dilution of 1:1000, a PCNA (Boster, CA, USA) at a dilution of 1:250, or an antibody against β-actin (Santa Cruz Biotechnology, Dallas, TX, US) at a dilution of 1:1000.

Techniques: Expressing, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of autologous bone marrow-derived stem cell mobilization on acute tubular necrosis and cell apoptosis in rats

doi: 10.3892/etm.2015.2592

Figure Lengend Snippet: Number of white blood cells in the peripheral blood (x10 9 /l) and the ratio of CD34 + cells among the mononuclear cells (%).

Article Snippet: Rabbit anti-rat CD34 + polyclonal antibody (dilution, 1:200; cat. no. bs-0754R; Beijing Bioss Biosynthesis Biotechnology Co., Ltd.) was used as the primary antibody for overnight incubation at 4°C.

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of autologous bone marrow-derived stem cell mobilization on acute tubular necrosis and cell apoptosis in rats

doi: 10.3892/etm.2015.2592

Figure Lengend Snippet: Number of cluster of differentiation 34+ cells in four groups of rat renal tissues (arrows indicate immunohistochemical staining; magnification, x200). (A–C) The (A) normal control, (B) treatment control and (C) model groups at day 5; (D–F) the model group at (D) day 10, (E) day 17 and (F) day 24; (G–J) the treatment group at (G) day 5, (H) day 10, (I) day 17 and (J) day 24.

Article Snippet: Rabbit anti-rat CD34 + polyclonal antibody (dilution, 1:200; cat. no. bs-0754R; Beijing Bioss Biosynthesis Biotechnology Co., Ltd.) was used as the primary antibody for overnight incubation at 4°C.

Techniques: Immunohistochemical staining, Staining