Journal: bioRxiv

Article Title: Follistatin-like protein 1 interacts with programmed cell death 4 to promote vascular mimicry in hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1101/2025.04.30.651401

Figure Lengend Snippet: High suppressor of Follistatin-like protein 1 (FSTL1) expression predicts poor prognosis in hepatocellular carcinoma (HCC). A Western blotting and RT-PCR analysis of FSTL1 protein expression in HCC (T) and non-tumor liver tissues (N). FSTL1 protein expression levels were normalized according to GAPDH expression levels (n = 8 per group). B Evidence of VM (red arrow) and angiogenesis (black arrow) in HCC samples (400x, scale bar=50μm). C. Immunohistochemical analysis of FSTL1 protein expression in human HCC tissues. D .HCC cells were transfected with FSTL1-Flag and subjected to immunoprecipitation using anti-Flag mAb. Co-immunoprecipitated PDCD4 was detected using anti-PDCD4 antibody (up panel).Endogenous FSTL1 in HCC cells was immunoprecipitated using anti-FSTL1 antibody with IgG as nonspecifc control (down panel). Co-immunoprecipitated PDCD4 was detected using anti-PDCD4 antibody.

Article Snippet: The details of the primary antibodies used are as follows: rabbit anti-FSTL1 (1:500, Proteintech, Rosemont, IL, USA), mouse anti-VIM (1:20,000, Proteintech), rabbit anti-PDCD4 (1:1,000, Abcam, Waltham, MA, USA), rabbit anti-E-cadherin (1:1,000, Abcam), rabbit anti-Twist1 (1:1,000, Abcam), rabbit anti-Snail (1:1,000, Abcam), rabbit anti-phospho-PI3K (1:1,000, Abcam), rabbit anti-PI3K (1:1,000, Abcam), rabbit anti-Akt (1:500, Abcam), rabbit anti-phospho-Akt (1:1,000, Abcam), rabbit anti-mTOR (1:2000, Abcam), and rabbit anti-phospho-mTOR (1:1,000, Abcam).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Transfection, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: Follistatin-like protein 1 interacts with programmed cell death 4 to promote vascular mimicry in hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1101/2025.04.30.651401

Figure Lengend Snippet: FSTL1 attenuates EMT of HCC cells by interacting with PDCD4. A. Western blot detected the expressions of FSTL1 in HCC cell lines. B. Western blot detected the expressions of FSTL1 hepG2 cells and MHCC97-H cells after the transfection .C,E. Proliferation ability of HCC cells was measured using CCK8 assays and colony formation assay after transfection. D. Transwell assay measured the migration and invasion ability of HCC cells. F. Western blot detects the expression of EMT-related proteins due to the interaction between FSTL1 and PDCD4. (E-cad, Vim and Snail-1).

Article Snippet: The details of the primary antibodies used are as follows: rabbit anti-FSTL1 (1:500, Proteintech, Rosemont, IL, USA), mouse anti-VIM (1:20,000, Proteintech), rabbit anti-PDCD4 (1:1,000, Abcam, Waltham, MA, USA), rabbit anti-E-cadherin (1:1,000, Abcam), rabbit anti-Twist1 (1:1,000, Abcam), rabbit anti-Snail (1:1,000, Abcam), rabbit anti-phospho-PI3K (1:1,000, Abcam), rabbit anti-PI3K (1:1,000, Abcam), rabbit anti-Akt (1:500, Abcam), rabbit anti-phospho-Akt (1:1,000, Abcam), rabbit anti-mTOR (1:2000, Abcam), and rabbit anti-phospho-mTOR (1:1,000, Abcam).

Techniques: Western Blot, Transfection, Colony Assay, Transwell Assay, Migration, Expressing

Journal: bioRxiv

Article Title: Follistatin-like protein 1 interacts with programmed cell death 4 to promote vascular mimicry in hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1101/2025.04.30.651401

Figure Lengend Snippet: FSTL1 attenuates HCC cell angiogenesis by interacting with PDCD4. A Matrigel-based tube formation assay assessed the angiogenesis ability. B Western blot detects the expression of angiogenesis related proteins due to the interaction between FSTL1 and PDCD4. (VEGF and HIF-1a).

Article Snippet: The details of the primary antibodies used are as follows: rabbit anti-FSTL1 (1:500, Proteintech, Rosemont, IL, USA), mouse anti-VIM (1:20,000, Proteintech), rabbit anti-PDCD4 (1:1,000, Abcam, Waltham, MA, USA), rabbit anti-E-cadherin (1:1,000, Abcam), rabbit anti-Twist1 (1:1,000, Abcam), rabbit anti-Snail (1:1,000, Abcam), rabbit anti-phospho-PI3K (1:1,000, Abcam), rabbit anti-PI3K (1:1,000, Abcam), rabbit anti-Akt (1:500, Abcam), rabbit anti-phospho-Akt (1:1,000, Abcam), rabbit anti-mTOR (1:2000, Abcam), and rabbit anti-phospho-mTOR (1:1,000, Abcam).

Techniques: Tube Formation Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Follistatin-like protein 1 interacts with programmed cell death 4 to promote vascular mimicry in hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1101/2025.04.30.651401

Figure Lengend Snippet: Effect of the interaction between FSTL1 and PDCD4 proteins on the expression of proteins related to the PI3K/Akt/mTOR pathway.

Article Snippet: The details of the primary antibodies used are as follows: rabbit anti-FSTL1 (1:500, Proteintech, Rosemont, IL, USA), mouse anti-VIM (1:20,000, Proteintech), rabbit anti-PDCD4 (1:1,000, Abcam, Waltham, MA, USA), rabbit anti-E-cadherin (1:1,000, Abcam), rabbit anti-Twist1 (1:1,000, Abcam), rabbit anti-Snail (1:1,000, Abcam), rabbit anti-phospho-PI3K (1:1,000, Abcam), rabbit anti-PI3K (1:1,000, Abcam), rabbit anti-Akt (1:500, Abcam), rabbit anti-phospho-Akt (1:1,000, Abcam), rabbit anti-mTOR (1:2000, Abcam), and rabbit anti-phospho-mTOR (1:1,000, Abcam).

Techniques: Expressing

Journal: bioRxiv

Article Title: Follistatin-like protein 1 interacts with programmed cell death 4 to promote vascular mimicry in hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1101/2025.04.30.651401

Figure Lengend Snippet: FSTL1 promotes the growth of transplanted tumors in nude mice by inhibiting PDCD4, regulates the expression of E-cad, vim, and snail, and activates the PI3K/Akt/mTOR pathway to promote VM formation in vivo. A-C .Tumor photos and tumor size throughout the experiment (22 days). D .Western blotting analyzed the impact of the interaction between FSTL1 and PDCD4 proteins on the expression of E-cad, vim, and snail proteins in tumor tissues, as well as the impact on PI3K/Akt/mTOR pathway-related proteins.E. Immunohistochemical analysis of the interaction between FSTL1 and PDCD4 proteins on the expression of angiogenesis-related proteins VEGF and CD31 in tumor tissues.

Article Snippet: The details of the primary antibodies used are as follows: rabbit anti-FSTL1 (1:500, Proteintech, Rosemont, IL, USA), mouse anti-VIM (1:20,000, Proteintech), rabbit anti-PDCD4 (1:1,000, Abcam, Waltham, MA, USA), rabbit anti-E-cadherin (1:1,000, Abcam), rabbit anti-Twist1 (1:1,000, Abcam), rabbit anti-Snail (1:1,000, Abcam), rabbit anti-phospho-PI3K (1:1,000, Abcam), rabbit anti-PI3K (1:1,000, Abcam), rabbit anti-Akt (1:500, Abcam), rabbit anti-phospho-Akt (1:1,000, Abcam), rabbit anti-mTOR (1:2000, Abcam), and rabbit anti-phospho-mTOR (1:1,000, Abcam).

Techniques: Expressing, In Vivo, Western Blot, Immunohistochemical staining

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: [ 68 Ga]Ga-NODAGA-RGD post MI reflects activated fibroblasts rather than angiogenesis

doi: 10.1007/s00259-025-07489-4

Figure Lengend Snippet: Violin-plot visualization of gene markers for injury-activated fibroblasts at day 5 post MI (Fstl1, Col1a1, Col3a1, Col5a1) comparing relative gene expression between the experimental groups and cell types. Median values are visualized by a black horizontal bar. ( n = 5 per group)

Article Snippet: Primary antibodies targeting FSTL1 (R&D Systems, AF1738, dilution 1:100), Integrin (Abcam, ab179473, dilution 1:100) CD31 (R&D Systems, AF3628, dilution 1:100) were applied overnight at 4°.

Techniques: Gene Expression

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: [ 68 Ga]Ga-NODAGA-RGD post MI reflects activated fibroblasts rather than angiogenesis

doi: 10.1007/s00259-025-07489-4

Figure Lengend Snippet: Expression pattern of α v β 3 -integrin, Fstl1 and CD31 in hearts at day 5 after permanent ligation of the LAD in both remote and infarcted myocardium, and the SHAM group. α v β 3 -integrin and Fstl1 are highly expressed within the infarct area ( A ), while profound less expression was detected in the remote area, comparable to the SHAM group ( B , C ). CD31 is expressed within the scar ( D ), but shows basal expression levels in the remote area ( E ) – similar to the SHAM group ( F ). Co-localization of Fstl1 and α v β 3 -integrin was quantified in co-labelled heart sections ( G ). Regions of Interest (ROI) were analyzed according to a positive fluorescence signal for both proteins ( H ). All α v β 3 -integrin positive ROIs were contained in the Fstl1-positive ROI population ( I ). Scale bar: 100 μm

Article Snippet: Primary antibodies targeting FSTL1 (R&D Systems, AF1738, dilution 1:100), Integrin (Abcam, ab179473, dilution 1:100) CD31 (R&D Systems, AF3628, dilution 1:100) were applied overnight at 4°.

Techniques: Expressing, Ligation, Fluorescence

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: Expression profiles of selected genes and proteins from microarray analysis, RT-PCR, immunocytochemistry and western blot analysis.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Expressing, Microarray, Immunocytochemistry, Western Blot

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: Validation of protein expression . Immunocytochemical analysis of protein expression of (a-f) Follistatin, (g-l) CD81, (m-r) AXL, Scale bars indicate 50 μm. (s) total protein expression analysis of the proteins Gas6 and CXCL1 comparing gliomas (N25, N29, N32) and NPC (HiB5, ST14A, RN33B). As well as RT-PCR analysis of activin expression in the tumors.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: Blocking antibody against the follistatin protein results in an increased tumor proliferation . Co-cultures of tumor N29 (A) and N32 (B) together with irradiated NPC (HiB5/ST14A), were treated by adding an antibody targeting the follistatin protein. Cells were seeded together and allowed to attach before addition of the antibodies. The plates were pulsed with 3 H-thymidine after 3 days in culture and cell division was measured as the amount of incorporated 3 H-thymidine. White bars show the proliferation of NPC + tumor control cultures and black bars show the proliferation after the addition of follistatin antibody. Mean ± stdev: Statistical analysis was made using t-test and Mann Whitney Wilcoxon rank sum test * P < 0.05.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Blocking Assay, Irradiation, Control, MANN-WHITNEY

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: A subpopulation of grade IV human GBM display follistatin expression . Figure 6. Follistatin expression by subsets of tumor cells within the glioblastoma multiforme (GBM) tumor cell bulk. Samples from GBM patients were freeze sectioned and subsequently stained using a follistatin antibody against the human antigen. A) Central tumor sections of patient A. A few selectively follistatin positive cells are seen within the central portions of the tumor. B) Central section from patient B. Follistatin expression is seen in a subset of tumor cells within the central bulk of the tumor. The histological examination of patient A and B tumors displays a heterogeneity within the tumor bulk with both positive and negative tumor cells. As negative control, the primary antibody was omitted, C. Scale bars indicate 50 μm.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Expressing, Staining, Negative Control

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: The validations of selected miRNAs to chicken TGFBR1 3’UTR. (A) The scheme of the constructed luciferase plasmid. (B) The relative luciferase expressions after miRNA mimic transfection. Transient transfection was conducted on 293T cells (3*10 4 cells/ well). The pmirGLO-WT-3’UTR plasmid (100 ng) was used, and co-treated with 5 nM miRNA mimics or siRNA (negative control, NC). Firefly and Renilla luminescence were detected after transfection for 24 hours. N= nine per group. Data were expressed as mean ± S.E.M. Transfection with pmirGLO-WT-3’UTR only was set as 1. All groups were compared with NC groups. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparisons test was used to evaluate differences between means (*P ≤ 0.05). (C) The sequence alignment of gga-miR-181a-5p and gga-miR-429-3p with the binding sites of the chicken TGFBR1 3’UTR.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Construct, Luciferase, Plasmid Preparation, Transfection, Negative Control, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: Target gene expressions after transient transfection for 48 hours using gga-miR-181a-5p or gga-miR-429-3p. The expressions of SOAT1 and TGFBR1 were analyzed by real-time PCR after 48 hours of miRNAs transfection. Data were expressed as mean ± S.E.M. N= nine per group. Control group was set as 1. All groups were compared with NC groups. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparisons test was used to evaluate differences between means (*P≤0.05).

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: TGFBR1 is one of the direct target gene of gga-miR-181a-5p and gga-miR-429-3p. The relative luciferase expressions after miRNA mimics transfection. (A) Scheme of potential binding sites of gga-miR-181a-5p and gga-miR-429-3p on the wild- or mutated-type of chicken TGFBR1 3’UTR. (B) The transient transfection was conducted on 293T cells (3*10 4 cells/ well). The pmirGLO-WT-3’UTR plasmid and pmirGLO-MU-3’UTR plasmid (100 ng/ well) were used and co-treated with 5 nM miRNA mimics. siRNA served as the negative control (NC). Firefly and Renilla luminescence were detected after transfection for 24 hours. N=10 to 14 per group. Data were expressed as mean ± S.E.M. All groups were compared with the NC group. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparison test was used to evaluate differences between means. A significant difference (*P≤0.05 or **P≤0.01) was indicated.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Luciferase, Transfection, Binding Assay, Plasmid Preparation, Negative Control

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: The SOAT1 and TGFBR1 protein levels after transfections with gga-miR-181a-5p or gga-miR-429-3p. EECs were transfected by miRNAs mimic for 48 hours and extracted for western blotting analysis. Total density of SOAT1 or TGFBR1 were normalized by total density of β-actin. Data were expressed as mean ± S.E.M. N = five to nine per group. C = group of no transfection in EECs, Mock = group of transfections with reagent only, NC = negative control group, the group of transfections with AllStars Negative Control siRNA. Others = groups of miRNAs transfections (5 nM or 30 nM miRNA mimics). Control group was set as 1. All groups were compared with NC groups. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparison test was used to evaluate differences between means. A significant difference (*P≤0.05 or **P≤0.01) was indicated.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Transfection, Western Blot, Negative Control

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: The possible relationship between miRNAs, TGFβ signaling pathway and SOAT1 expressions. The TGFβ signaling pathway is activated when the ligand (e.g., TGFβ1) binds to TGFBR2, TGFBR2 phosphorylates TGFBR1. The signal transmitter Smad2/3 is phosphorylated by TGFBR1 and joins with phosphorylated Smad4 to form SMAD complex in cytoplasm. The SMAD complex then enters nucleus to target to transcription factor binding site to affect SOAT1 gene expression. However, the gga-miR-181a-5p and gga-miR-429-3p both have the inhibitory ability on TGFBR1, and then decrease the SOAT1 expression. Although the gga-miR-133a-5p is found to attenuate SOAT1 expression, the effect is independent of the TGFβ signaling pathway.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Binding Assay, Expressing

Journal: Cells

Article Title: Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage.

doi: 10.3390/cells12050801

Figure Lengend Snippet: Figure 1. Localization of the follistatin protein in normal rat kidneys. (a) Localization of follistatin (brown) in normal rat kidneys was examined by immunostaining. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 0.2 mm. (b–f) Identification of follistatin-positive tubules in rat normal kidneys using serial sections. Aquaporin 1, AQP1; Na-Cl co-transporter, NCC; aquaporin 2, AQP2. Asterisks indicate identical tubules. Bar = 50 µm.

Article Snippet: The primary antibodies used in this study are as follows: mouse anti-follistatin antibody (60060-1-ig; Proteintech, Rosemont, IL, USA), rabbit anti-inhibin βA antibody (ab97705; Abcam, Cambridge, UK), rabbit anti-Na-Cl co-transporter (NCC) antibody (ab3553; Abcam), mouse anti-aquaporin (AQP)-1 antibody (sc-25287), mouse anti- aquaporin (AQP)-2 antibody (sc-515770), mouse anti-megalin (G-9) antibody (sc-515750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-uromodulin antibody (HPA043420; Sigma-Aldrich, St. Louis, MO, USA), goat anti-kidney injury molecule (KIM)-1 anti- Cells 2023, 12, 801 3 of 12 body (AF3689), and goat anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (AF1857; R&D Systems, Minneapolis, MN, USA).

Techniques: Immunostaining

Journal: Cells

Article Title: Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage.

doi: 10.3390/cells12050801

Figure Lengend Snippet: Figure 2. Localization of the follistatin protein in kidneys after renal ischemia. (a) Localization of follistatin (brown) in kidneys after renal ischemia was examined by immunostaining (left panel). Bar = 2 mm. Quantitative analysis of follistatin-positive area (right panel). Five randomly selected fields of the kidney were assessed at ×100 magnification. The follistatin-positive area was measured by ImageJ. Values are the means ± S.E. (n = 6–8). * p < 0.05, ** p < 0.01 vs. 0 h (b,c) Localization of the follistatin protein in normal and ischemic kidneys. The kidneys were collected at 0 h and 24 h after reperfusion. Ischemia reperfusion, IR. Bar = 0.2 mm in (b), 50 µm in (c). Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 50 µm. (d) Localization of follistatin protein and megalin, aquaporin 1 (AQP1), uromodulin (UMOD), or aquaporin 2 (AQP2) in ischemic kidneys was evaluated using serial sections. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 50 µm. (e) Localization of follistatin, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) in ischemic kidneys was evaluated using serial sections. Bar = 0.2 mm (upper panels), 50 µm (lower panels). (f) Localization of follistatin and activin A in ischemic kidneys was evaluated using serial sections. Bar = 50 µm. Asterisks indicate identical tubules.

Article Snippet: The primary antibodies used in this study are as follows: mouse anti-follistatin antibody (60060-1-ig; Proteintech, Rosemont, IL, USA), rabbit anti-inhibin βA antibody (ab97705; Abcam, Cambridge, UK), rabbit anti-Na-Cl co-transporter (NCC) antibody (ab3553; Abcam), mouse anti-aquaporin (AQP)-1 antibody (sc-25287), mouse anti- aquaporin (AQP)-2 antibody (sc-515770), mouse anti-megalin (G-9) antibody (sc-515750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-uromodulin antibody (HPA043420; Sigma-Aldrich, St. Louis, MO, USA), goat anti-kidney injury molecule (KIM)-1 anti- Cells 2023, 12, 801 3 of 12 body (AF3689), and goat anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (AF1857; R&D Systems, Minneapolis, MN, USA).

Techniques: Immunostaining

Journal: Cells

Article Title: Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage.

doi: 10.3390/cells12050801

Figure Lengend Snippet: Figure 3. Expression and localization of follistatin mRNA in normal and ischemic rat kidneys. (a) Localization of follistatin mRNA in normal kidneys was examined by in situ hybridization. Hybridization signals are shown as blue color. Bar = 0.2 mm (left panel), 50 µm (right panels). (b) Follistatin mRNA (blue) and uromodulin (brown) in normal kidneys was evaluated by double staining. Cortex, CO; outer medulla, OM; inner medulla, IM. Bar = 0.2 mm (left panel), 50 µm (right panels). (c) Localization of follistatin mRNA and aquaporin 1 (AQP1) in normal rat kidneys was evaluated using serial sections. Asterisks indicate identical tubules. (d) Follistatin mRNA (blue) and uromodulin (brown) in normal and ischemic kidneys at 24 after reperfusion was evaluated by double staining. Cortex, CO; outer medulla, OM. (e) Follistatin mRNA (blue) and aquaporin 2 (AQP2, brown) in normal and ischemic kidneys was evaluated by double staining. Inner medulla, IM. Bar = 50 µm in (c–e). (f) Quantitative analysis of follistatin mRNA expression by real-time PCR. Values (relative expression ratio to GAPDH) are means ± S.E. (n = 5–8).

Article Snippet: The primary antibodies used in this study are as follows: mouse anti-follistatin antibody (60060-1-ig; Proteintech, Rosemont, IL, USA), rabbit anti-inhibin βA antibody (ab97705; Abcam, Cambridge, UK), rabbit anti-Na-Cl co-transporter (NCC) antibody (ab3553; Abcam), mouse anti-aquaporin (AQP)-1 antibody (sc-25287), mouse anti- aquaporin (AQP)-2 antibody (sc-515770), mouse anti-megalin (G-9) antibody (sc-515750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-uromodulin antibody (HPA043420; Sigma-Aldrich, St. Louis, MO, USA), goat anti-kidney injury molecule (KIM)-1 anti- Cells 2023, 12, 801 3 of 12 body (AF3689), and goat anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (AF1857; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, In Situ Hybridization, Hybridization, Double Staining, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage.

doi: 10.3390/cells12050801

Figure Lengend Snippet: Figure 4. Urinary follistatin levels significantly increased after renal ischemia. (a) Time course changes in urinary follistatin and serum creatinine levels after reperfusion (n = 8). (b) Urinary and serum follistatin levels in normal and renal ischemia (45 min) rats were measured by ELISA. Values are means ± S.E. (n = 8). *** p < 0.001; N.S., not significant. (c) Time course changes in urinary follistatin, urinary NGAL, KIM-1, and L-FABP after reperfusion. (d,e) Correlations between urinary follistatin and urinary NGAL (d) or urinary KIM-1 (e) at 24 h after reperfusion. Renal ischemia (45 min) was performed, and urine was collected 24 h after reperfusion.

Article Snippet: The primary antibodies used in this study are as follows: mouse anti-follistatin antibody (60060-1-ig; Proteintech, Rosemont, IL, USA), rabbit anti-inhibin βA antibody (ab97705; Abcam, Cambridge, UK), rabbit anti-Na-Cl co-transporter (NCC) antibody (ab3553; Abcam), mouse anti-aquaporin (AQP)-1 antibody (sc-25287), mouse anti- aquaporin (AQP)-2 antibody (sc-515770), mouse anti-megalin (G-9) antibody (sc-515750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-uromodulin antibody (HPA043420; Sigma-Aldrich, St. Louis, MO, USA), goat anti-kidney injury molecule (KIM)-1 anti- Cells 2023, 12, 801 3 of 12 body (AF3689), and goat anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (AF1857; R&D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage.

doi: 10.3390/cells12050801

Figure Lengend Snippet: Figure 5. Correlation between urinary follistatin and severity of kidney damage. (a,b) Serum creatinine and BUN in rats with renal ischemia at 24 h after reperfusion. N.S., not significant, * p < 0.05, *** p < 0.001 vs. 0 h. (c–e) Urinary follistatin, urinary NGAL, and urinary KIM-1 in rats with renal ischemia for the indicated periods at 24 h after reperfusion. N.S., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 h. (f) Localization of follistatin (brown) in kidneys after renal ischemia for 15, 30, and 45 min was examined by immunostaining. Bar = 2 mm. (g) Quantitative analysis of follistatin-positive area. Five randomly selected fields of the kidney were assessed at ×100 magnification. The follistatin-positive area was measured by ImageJ 1.53a. Values are the means ± S.E. (n = 5–6). N.S., not significant, * p < 0.05 vs. 0 h. (h) Semiquantitative analysis of the histological changes induced by renal ischemia. The ATN area was calculated as described in Section 2. N.S., not significant, * p < 0.05, ** p < 0.01 vs. 0 h. (i,j). Correlation between urinary follistatin and follistatin-positive area (i) or ATN area (j) at 24 h after reperfusion.

Article Snippet: The primary antibodies used in this study are as follows: mouse anti-follistatin antibody (60060-1-ig; Proteintech, Rosemont, IL, USA), rabbit anti-inhibin βA antibody (ab97705; Abcam, Cambridge, UK), rabbit anti-Na-Cl co-transporter (NCC) antibody (ab3553; Abcam), mouse anti-aquaporin (AQP)-1 antibody (sc-25287), mouse anti- aquaporin (AQP)-2 antibody (sc-515770), mouse anti-megalin (G-9) antibody (sc-515750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-uromodulin antibody (HPA043420; Sigma-Aldrich, St. Louis, MO, USA), goat anti-kidney injury molecule (KIM)-1 anti- Cells 2023, 12, 801 3 of 12 body (AF3689), and goat anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (AF1857; R&D Systems, Minneapolis, MN, USA).

Techniques: Immunostaining

Journal: Cells

Article Title: Tubule-Derived Follistatin Is Increased in the Urine of Rats with Renal Ischemia and Reflects the Severity of Acute Tubular Damage.

doi: 10.3390/cells12050801

Figure Lengend Snippet: Figure 6. Possible mechanism of follistatin excretion into urine after renal ischemia. Aquaporin 1, AQP1; aquaporin 2, AQP2; Na-Cl co-transporter, NCC.

Article Snippet: The primary antibodies used in this study are as follows: mouse anti-follistatin antibody (60060-1-ig; Proteintech, Rosemont, IL, USA), rabbit anti-inhibin βA antibody (ab97705; Abcam, Cambridge, UK), rabbit anti-Na-Cl co-transporter (NCC) antibody (ab3553; Abcam), mouse anti-aquaporin (AQP)-1 antibody (sc-25287), mouse anti- aquaporin (AQP)-2 antibody (sc-515770), mouse anti-megalin (G-9) antibody (sc-515750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-uromodulin antibody (HPA043420; Sigma-Aldrich, St. Louis, MO, USA), goat anti-kidney injury molecule (KIM)-1 anti- Cells 2023, 12, 801 3 of 12 body (AF3689), and goat anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (AF1857; R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-4458 inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells by suppressing the TGF-β signaling pathway via targeting TGFBR1.

doi: 10.1093/abbs/gmaa029

Figure Lengend Snippet: Figure 4. miR-4458 targets TGFBR1 and suppresses the TGF-β signaling pathway (A) Venn diagram of common target genes from four databases including TargetScan, DIANA TOOLS, miRWalk, and miRDB. (B) The predicted complementary binding sites of miR-4458 and TGFBR1. (C) Overexpression of miR-4458 reduced the luciferase activity of Wt TGFBR1 3′-UTR. However, upregulated miR-4458 showed no effect on the Mut TGFBR1 3′-UTR. (D,E) The mRNA expression of TGFBR1 after transfection with miR-4458 mimics or inhibitor in HCC cells. (F,G) Western blot analysis revealed that overexpression of miR-4458 reduced the protein expression of TGFBR1 and p-Smad2/3. Data are presented as the mean±SD. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001.

Article Snippet: The following primary antibodies were used: anti-TGFBR1 antibody (Abcam, Cambridge, USA), anti-E-cadherin antibody (Affinity, Piscataway, USA), anti-N-cadherin antibody (Cell Signaling Technology, Danvers, USA), anti-p-Smad2/3 antibody (Cell Signaling Technology), anti-Vimentin antibody (Bimake), anti-Snail antibody (Wanleibio, Shenyang, China), and anti-GAPDH antibody (Beyotime).

Techniques: Binding Assay, Over Expression, Luciferase, Activity Assay, Expressing, Transfection, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-4458 inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells by suppressing the TGF-β signaling pathway via targeting TGFBR1.

doi: 10.1093/abbs/gmaa029

Figure Lengend Snippet: Figure 5. Overexpression of TGFBR1 reverses the inhibitory effect of miR-4458 in Hep3B cells (A,B) The miR-4458 mimics and TGFBR1 overexpression plasmid were cotransfected into Hep3B cells. Wound healing and transwell assays were used to analyze the migration and invasion ability of Hep3B cells. (C) The protein expressions of E-cadherin, N-cadherin, Vimentin, and Snail were detected after cotransfection with the miR-4458 mimics and TGFBR1 overexpression plasmid. (D) Western blot analysis was used to evaluate the expressions of TGFBR1 and p-Smad2/3 after cotransfection with the miR-4458 mimics and TGFBR1 overexpression plasmid. Data are presented as the mean±SD. ∗P<0.05, ∗∗P<0.01.

Article Snippet: The following primary antibodies were used: anti-TGFBR1 antibody (Abcam, Cambridge, USA), anti-E-cadherin antibody (Affinity, Piscataway, USA), anti-N-cadherin antibody (Cell Signaling Technology, Danvers, USA), anti-p-Smad2/3 antibody (Cell Signaling Technology), anti-Vimentin antibody (Bimake), anti-Snail antibody (Wanleibio, Shenyang, China), and anti-GAPDH antibody (Beyotime).

Techniques: Over Expression, Plasmid Preparation, Migration, Cotransfection, Western Blot

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 1. A, Plasma follistatin in relation to 3 h of bicycling exercise (n 5). The shaded area marks the 3 h period of exercise. B, Arterial and venous follistatin plasma concentrations during 2 h of one-legged knee-extensor exercise and the following recovery period (n 9). The shaded area marks the 2-h period of exercise. C, Follistatin mRNA expression in skeletal muscle. D, Follistatin mRNA expression in C2C12 muscle cells in response to EPS. E, Follistatin protein release from C2C12 muscle cells in response to EPS. *, P 0.05.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Clinical Proteomics, Expressing

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 2. A, Plasma follistatin in mice in relation to 1 h of swimming and the following recovery period (n 8 at each time point). B, Regulation of follistatin mRNA expression in mice in relation to 1 h of swimming and the following recovery period (n 8 at each time-point). The following tissues were analyzed: spleen, liver, subcutaneous (SubQ) and visceral fat (Vis), soleus, and gastrocnemius (Gastro) muscle, heart, and kidney. C, Western blot on liver tissue from mice performing 1 h of swimming exercise. Time points 1 h, 3 h, and 6 h are depicted as the relative proportion of the 0 h levels. *, P 0.05.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Clinical Proteomics, Expressing, Western Blot

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 4. A, Injection (ip) of 2 mg/kg epinephrine to mice (n 10 in each group). Plasma glucose before and after epinephrine injection. B, Injection (ip) of 2 mg/kg epinephrine to mice (n 10 in each group) Expression of follistatin in the liver in the control and epinephrine- stimulated groups. *, P 0.05. C, Hepatocytes treated with EPS- conditioned media from C2C12 myotubes. CON, Media from non-EPS- stimulated myotubes; MO, negative control consisting of media only; POS, TGF- were used as a positive control. Only TGF- could induce an increase in follistatin mRNA expression.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Injection, Clinical Proteomics, Expressing, Control, Negative Control, Positive Control

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 3. A, Infusion of recombinant IL-6 in healthy young men (n 7). A, Plasma IL-6 during a 3-h IL-6-infusion study. The shaded area marks the 3-h period of IL-6 infusion. B, Plasma follistatin during the 3-h IL-6- infusion study. No regulation of plasma follistatin. *, P 0.05. The shaded area marks the 3-h period of IL-6 infusion.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Recombinant, Clinical Proteomics