Journal: Journal of Thoracic Disease

Article Title: Exacerbation of pulmonary fibrosis following acute lung injury via activin-A production by recruited alveolar macrophages

doi: 10.21037/jtd-24-680

Figure Lengend Snippet: The impact of activin-A on MLE-12 cells. (A) Representative images of coimmunostaining (IF staining, 400×) for ALK4 (green) and E-cadherin (red) in lung tissue slides from each group of mice. White arrows: epithelial cells co-expressing ALK4 and E-cadherin. (B) E-cadherin, Vimentin, and α-SMA expressions in the MLE-12 cells with 7 days of activin-A treatment. Left panel: representative Western blotting. (C) The EMT process was examined via IF staining in activin-A treated MLE-12 cells. (D) Left panel: Wound-healing assay for measurement of MLE-12 cell migration with activin-A treatment. Right panel: bar graph showing the mean migration rate with 12- and 24-h stimulation. (E) The expression of E-cadherin, Vimentin, and α-SMA expression in the MLE-12 cells with 7 days of SLM treatment. Upper panel: representative Western blotting. (F,G) Increased phosphorylation of SMAD2/3 after 24-h activin-A or SLM stimulation. Upper panel: representative Western blotting. Quantitative results are expressed as mean ± standard error of the mean. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. +/−: with/without the stimulation of activin-A/FST. ns, no significance; ALK4, activin receptor-like kinase 4; LPS, lipopolysaccharide; α-SMA, alpha smooth muscle actin; EMT, epithelial-mesenchymal transition; FST, follistatin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SLM, supernatants collected from LPS-treated macrophages; p-SMAD2/3, phosphorylated SMAD2/3; SMAD, suppressor of mothers against decapentaplegic; MLE-12, murine lung epithelial 12; IF, immunofluorescence.

Article Snippet: Recombinant activin-A (50 μg; HY-P70311) and the follistatin (FST; HY-P74148; 50 μg) proteins were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Staining, Expressing, Western Blot, Wound Healing Assay, Migration, Phospho-proteomics, Immunofluorescence

Journal: Journal of Thoracic Disease

Article Title: Exacerbation of pulmonary fibrosis following acute lung injury via activin-A production by recruited alveolar macrophages

doi: 10.21037/jtd-24-680

Figure Lengend Snippet: The impact of activin-A on TGF-β and Hippo signaling pathway in BMDMs. (A) Activin-A promoted SMAD2 phosphorylation. Left panel: the expression of p-SMAD2 and SMAD2 after 2-h activin-A stimulation. (B) Activin-A promoted p-YAP and p-TAZ dephosphorylation, as well as the nuclear accumulation of SMAD2, YAP, and TAZ. Left panel: the expression and location of p-SMAD2, p-YAP, p-TAZ, SMAD2, YAP, and TAZ after 2-h activin-A stimulation. (C) IF staining detected the nuclear translocation of SMAD2, YAP, and TAZ in activin-A-treated BMDMs; the representative images were taken at 630× magnification. (D) The expression of p-SMAD2 after 2-h activin-A stimulation. Silencing of TAZ blunted activin-A-induced p-SMAD2 expression. (E) The expression of p-SMAD2 after 2-h activin-A stimulation. Silencing of YAP decreased activin-A-induced SMAD2 phosphorylation. (F) Overexpression of TAZ promoted the activin-A-induced p-SMAD2 expression. (G) Overexpression of YAP increased the levels of p-SMAD2 under activin-A treatment. (H) Overexpression of TAZ and YAP promoted the expression of Fn1 . (I) Yap or Taz deficiency reduced Inhba mRNA quantity. (J) The overexpression of Yap or Taz increased the mRNA levels of Inhba . Quantitative results are presented as mean ± standard error of the mean. *, P<0.05; **, P<0.01; ***, P<0.001. +/−: with/without the stimulation of activin-A. ns, no significance; α-SMA, alpha smooth muscle actin; BMDM, bone marrow-derived macrophage; FST, follistatin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; PBS, phosphate-buffered saline; p-SMAD2, phosphorylated SMAD2; SMAD, suppressor of mothers against decapentaplegic; TAZ, transcriptional coactivated with PDZ-binding motif; TGF-β, transforming growth factor-β; YAP, yes-associated protein.

Article Snippet: Recombinant activin-A (50 μg; HY-P70311) and the follistatin (FST; HY-P74148; 50 μg) proteins were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Phospho-proteomics, Expressing, De-Phosphorylation Assay, Staining, Translocation Assay, Over Expression, Derivative Assay, Negative Control, Saline, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: Exacerbation of pulmonary fibrosis following acute lung injury via activin-A production by recruited alveolar macrophages

doi: 10.21037/jtd-24-680

Figure Lengend Snippet: Effect of FST on postinjury lung fibrosis. (A) Images from H&E staining (100×) in lung tissue slides at day 7 after LPS challenge and the semiquantitative lung injury scores. (B,C) Collagen deposition as measured by Masson (100×) and Sirius red staining (100×). (D) Abundance of α-SMA as detected by IF staining (400×). Quantitative data are expressed as mean ± standard error of the mean. *, P<0.05; **, P<0.01; ****, P<0.0001. α-SMA, alpha smooth muscle actin; FST, follistatin; H&E, hematoxylin and eosin; IF, immunofluorescence; LPS, lipopolysaccharide.

Article Snippet: Recombinant activin-A (50 μg; HY-P70311) and the follistatin (FST; HY-P74148; 50 μg) proteins were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Staining, Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Screening and Identification of Differential Ovarian Proteins before and after Induced Ovulation via Seminal Plasma in Bactrian Camels

doi: 10.3390/ani11123512

Figure Lengend Snippet: Primer sequences of the mouse genes used for qPCR.

Article Snippet: The membranes were blocked for 2 h in Tris–HCl buffer containing 5% ( w / v ) non-fat milk powder, then were incubated overnight at 4 °C with the following primary antibodies from mice or rabbits: ACTB (1:5000, Proteintech, China), CAMK2G (1:1000, Proteintech), FST (1:1000, Proteintech), NR5A1 (1:500, Proteintech), PAK1 (1:1000, Santa Cruz, USA), PRL (1:1000, Santa Cruz), STAG2 (1:1000, Santa Cruz), TGFB2 (1:500, Santa Cruz), Wnt2b (1:500, Santa Cruz), GAPDH (1:5000, Proteintech).

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Screening and Identification of Differential Ovarian Proteins before and after Induced Ovulation via Seminal Plasma in Bactrian Camels

doi: 10.3390/ani11123512

Figure Lengend Snippet: Differentially expressed proteins in the ovarian tissues of Bactrian camels after SP-induced ovulation.

Article Snippet: The membranes were blocked for 2 h in Tris–HCl buffer containing 5% ( w / v ) non-fat milk powder, then were incubated overnight at 4 °C with the following primary antibodies from mice or rabbits: ACTB (1:5000, Proteintech, China), CAMK2G (1:1000, Proteintech), FST (1:1000, Proteintech), NR5A1 (1:500, Proteintech), PAK1 (1:1000, Santa Cruz, USA), PRL (1:1000, Santa Cruz), STAG2 (1:1000, Santa Cruz), TGFB2 (1:500, Santa Cruz), Wnt2b (1:500, Santa Cruz), GAPDH (1:5000, Proteintech).

Techniques: Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Screening and Identification of Differential Ovarian Proteins before and after Induced Ovulation via Seminal Plasma in Bactrian Camels

doi: 10.3390/ani11123512

Figure Lengend Snippet: Validation of differentially expressed proteins (DEPs). ( A ) Protein expression levels detected in ovarian protein extracts from Bactrian camels via iTRAQ LC-MS/MS. ( B ) Western blot analysis of PAK1, PRL, STAG2, TGFB2, CAMK2G, FST, NR5A1, and Wnt2b in ovarian protein extracts from Bactrian camels. ( C ) Integrated optical density analysis of the Western blots using ImageJ. Data are presented as the mean ± standard deviation; * p < 0.05, ** p < 0.01. ( D ) Venn diagram of the DEPs using multiple comparisons of the endocrine system, reproductive system and neural signal transduction system groups. ( E ) Immunofluorescence staining to localize the expression patterns of the DEPs in the ovarian samples.

Article Snippet: The membranes were blocked for 2 h in Tris–HCl buffer containing 5% ( w / v ) non-fat milk powder, then were incubated overnight at 4 °C with the following primary antibodies from mice or rabbits: ACTB (1:5000, Proteintech, China), CAMK2G (1:1000, Proteintech), FST (1:1000, Proteintech), NR5A1 (1:500, Proteintech), PAK1 (1:1000, Santa Cruz, USA), PRL (1:1000, Santa Cruz), STAG2 (1:1000, Santa Cruz), TGFB2 (1:500, Santa Cruz), Wnt2b (1:500, Santa Cruz), GAPDH (1:5000, Proteintech).

Techniques: Biomarker Discovery, Expressing, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Western Blot, Standard Deviation, Transduction, Immunofluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Screening and Identification of Differential Ovarian Proteins before and after Induced Ovulation via Seminal Plasma in Bactrian Camels

doi: 10.3390/ani11123512

Figure Lengend Snippet: Expression and localization analysis of the differentially expressed proteins (DEPs) in the hypothalamic–pituitary–ovarian axis. ( A ) FST, NR5A1 and PRL mRNA expression levels in Bactrian camel tissues detected via RT-PCR. ( B ) Optical density was analyzed via PCR and ImageJ. Different letters indicate significant differences within groups. ( C ) Western blot analysis of FST, NR5A1 and PRL proteins in different tissue samples. ( D ) Optical density value was analyzed via ImageJ.

Article Snippet: The membranes were blocked for 2 h in Tris–HCl buffer containing 5% ( w / v ) non-fat milk powder, then were incubated overnight at 4 °C with the following primary antibodies from mice or rabbits: ACTB (1:5000, Proteintech, China), CAMK2G (1:1000, Proteintech), FST (1:1000, Proteintech), NR5A1 (1:500, Proteintech), PAK1 (1:1000, Santa Cruz, USA), PRL (1:1000, Santa Cruz), STAG2 (1:1000, Santa Cruz), TGFB2 (1:500, Santa Cruz), Wnt2b (1:500, Santa Cruz), GAPDH (1:5000, Proteintech).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Animals : an Open Access Journal from MDPI

Article Title: Screening and Identification of Differential Ovarian Proteins before and after Induced Ovulation via Seminal Plasma in Bactrian Camels

doi: 10.3390/ani11123512

Figure Lengend Snippet: ( A ) Representative images of hematoxylin and eosin staining of the hypothalamus, pituitary, pineal gland, ovaries, oviduct, and uterus. ( B ) Intracellular localization analysis of FST in different tissues. ( C ) Intracellular localization analysis of NR5A1 in different tissues. ( D ) Intracellular localization analysis of PRL in different tissues.

Article Snippet: The membranes were blocked for 2 h in Tris–HCl buffer containing 5% ( w / v ) non-fat milk powder, then were incubated overnight at 4 °C with the following primary antibodies from mice or rabbits: ACTB (1:5000, Proteintech, China), CAMK2G (1:1000, Proteintech), FST (1:1000, Proteintech), NR5A1 (1:500, Proteintech), PAK1 (1:1000, Santa Cruz, USA), PRL (1:1000, Santa Cruz), STAG2 (1:1000, Santa Cruz), TGFB2 (1:500, Santa Cruz), Wnt2b (1:500, Santa Cruz), GAPDH (1:5000, Proteintech).

Techniques: Staining