Journal: Journal of cachexia, sarcopenia and muscle

Article Title: The Innovative Role of Nuclear Receptor Interaction Protein in Orchestrating Invadosome Formation for Myoblast Fusion.

doi: 10.1002/jcsm.13598

Figure Lengend Snippet: FIGURE 1 | NRIP is an actin-binding protein. (A) NRIP directly interacted with actin in vitro. Upper panel: His-MBP and His-MBP-NRIP proteins were examined using Coomassie blue staining. The arrows indicate His-MBP and His-MBP-NRIP. Lower panel: GST and GST-actin proteins indicated by arrows. (B) Left panel: His pull-down assy. Right panel: GST pull-down assay. The pull-downed extracts were analysed by western blotting with an anti-His antibody to detect His-MBP and His-MBP-NRIP and an anti-GST antibody to detect GST-actin. (C) NRIP interacted with actin in C2C12 myotubes. The protein extracts from C2C12 myotubes were immunoprecipitated with anti-NRIP or anti-alpha-actin and immunoblotted with indicated antibodies. The rabbit normal IgG (rIgG) served as the antibody control for anti-NRIP, while the mouse normal IgG (mIgG) was the control for anti-alpha-actin. (D) NRIP interacted with actin in cells. Cell extracts (1 mg) from 293T cells co-transfected with Flag-NRIP and mCherry-actin were immunoprecipitated with anti-mCherry (actin) (left panel) or with anti-Flag (NRIP) (right panel) and immunoblotted with indicated antibodies. (E) Subcellular localization of NRIP in C2C12 cells. Nuclear (N), cytosolic (C), and membrane (M) fractions from C2C12 cells were extracted as described in Methods. EGFR was a positive control of membrane proteins, PCNA was a positive control for nuclear proteins, and GAPDH was a positive control for cytosolic proteins. % of total NRIP was determined as the intensity ratio of nuclear, cytosolic, or membrane NRIP to total NRIP (N = 3). (F) Endogenous NRIP was located at the plasma membrane, nucleus and cytoplasm. C2C12 myoblasts were differentiated for 4 days and stained with anti-NRIP (green), phalloidin for the cell membrane (red, F-actin staining), and DAPI for the nucleus (blue). Arrowheads: either NRIP or F-actin at the plasma membrane. Arrows: NRIP in the cytoplasm. Asterisks: NRIP in the nucleus. Scale bar: 20 μm.

Article Snippet: Protein lysates (50 μg) were subjected to western blotting with indicated primary antibodies: anti- NRIP (A302- 434A, Novus, 1:2000), anti- EGF receptor (#2646, Cell Signalling, 1:1000), anti- DsRed (632496, TaKaRa, 1:10 000), anti- actin (ab179467, Abcam, 1:10 000), anti- EGFP (ab6556, Abcam, 1:10 000), anti- flag (ab1162, Abcam, 1:10 000), anti- MyHC (ab124205, Abcam, 1:2000), anti- Tks5 (sc- 376211, Santa Cruz, 1:1000), anti- cortactin (sc55579, Santa Cruz, 1:1000), anti- His (66005- 1- Ig, Proteintech, 1:10 000), anti- GST (sc- 459, Santa Cruz, 1:10 000), anti- PCNA (#2586, Cell Signalling, 1:10 000), and anti- GAPDH (LFPA0212, AbFrontier, 1:10 000).

Techniques: Binding Assay, In Vitro, Staining, Pull Down Assay, Western Blot, Immunoprecipitation, Control, Transfection, Membrane, Positive Control, Clinical Proteomics

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