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: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Up-regulation of Fstl1 in silica-injured mice and patients with silicosis. ( a ) Fstl1 mRNA expression in lung tissues of C57BL/6J mice at the different time-point after silica injury was determined by qRT-PCR analysis (n = 6 per group; * P < 0.05; ** P < 0.01 by one-way ANOVA followed by Student’s t test). ( b ) FSTL1 protein in lung tissues of C57BL/6J mice at the different time-point after silica injury was determined by western blot analysis. β-tubulin was used as a loading control. ( c ) Immunohistochemistry (IHC) of FSTL1 in lung sections of C57BL/6J mice 21 days after saline or silica injury. Representative images of the staining are shown. NA stands for normal area, FA stands for fibrotic area; both are shown at higher magnification. (n = 6 per group; scale bars, 200 μm). ( d ) Fstl1 mRNA expression in primary alveolar macrophages (AMs), alveolar epithelial cells (AECs) and fibroblasts (Fb) at day 21 after saline or silica injury was determined by qRT-PCR analysis (n = 6 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( e ) FSTL1 protein in serum of C57BL/6J mice at the different time-point after silica injury was determined by ELISA analysis (n = 6 per group; * P < 0.05; ** P < 0.01 by one-way ANOVA followed by Student’s t test). ( f ) Representative images of lung fibrotic area of patient with silicosis with H&E staining and immunohistochemical staining for FSTL1 protein (NA stands for normal area, FA stands for fibrotic area; Scale bars, 200 μm). ( g ) FSTL1 levels in serum of patients with silicosis and normal control individuals were determined by ELISA (* P < 0.05 by one-way ANOVA followed by Student’s t test).

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemistry, Saline, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Fstl1 +/− mice have an attenuated pulmonary fibrosis phenotype. ( a ) Western blot analysis of FSTL1 expression in lung tissues of Fstl1 +/− and WT mice 21 days after saline or silica injury. β-tubulin was used as a loading control. ( b ) H&E staining of lung sections of Fstl1 +/− and WT mice 21 days after silica injury. Representative images of the staining are shown. Arrows show inflammatory cells. (n = 6 per group; scale bars, 200 μm). ( c ) Lung fibrotic score analysis of the lung sections from Fstl1 +/− and WT mice 21 days after silica injury (n = 6 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). The fibrotic area is presented as a percentage. ( d ) Hydroxyproline contents in lung tissues from Fstl1 +/− and WT mice were measured 21 days after saline or silica injury (n = 6 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( e ) Masson trichrome staining of lung sections of Fstl1 +/− and WT mice 21 days after silica treatment. Representative images of the staining are shown (n = 6 per group; scale bars, 200 μm). ( f ) qRT-PCR analysis of Col1a1 mRNA expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica injury (n = 3 per group; *** P < 0.001 by one-way ANOVA followed by Student’s t test). ( g ) Western blot analysis of type I collagen (Col1) expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica injury (n = 3 per group). β-tubulin was used as a loading control. ( h ) qRT-PCR analysis of fibronectin ( Fn1 ) mRNA expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica injury (n = 3 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test).

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Western Blot, Expressing, Saline, Control, Staining, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Fstl1 +/− mice have an attenuated pulmonary inflammatory response. ( a–e ) Fstl1 +/− and their WT littermate mice were intratracheally exposured to saline or 200 mg/Kg silica, bronchoalveolar lavage fluid (BALF) were collected from Fstl1 +/− and WT mice 7 days after administration of saline or silica. ( a ) The number of total BALF cells was determined by hemocytometer (n = 7 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( b–d ) The differential cell counts in BALF were determined according to standard morphologic criteria. ( b ) Macrophages (n = 7 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( c ) PMNs (Polymorphonuclear neutrophils, n = 7 per group). ( d ) Lymphocytes (n = 7 per group). ( e ) The level of cytokine IL-1β in BALF was detected by ELISA assay (n = 7 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( f–g ) Fstl1 +/− and their WT littermate mice were intratracheally exposured to saline or silica, lung tissues were collected from Fstl1 +/− and WT mice 7 days after administration of saline or silica. ( f ) The level of cytokine IL-1β in lung tissues was detected by ELISA assay (n = 4 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( g ) The levels of NLRP3 and caspase-1 (p20) in lung tissues were determined by western blot analysis. β-tubulin was used as a loading control.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Fstl1 +/− mice have less myofibroblast accumulation after silica exposure. ( a ) Immunofluorescence analysis of α-SMA expression in lung sections of Fstl1 +/− and WT mice 21 days after saline or silica exposure. Representative images of the staining are shown. (α-SMA, green; Endomucin, red; nucleus, blue; scale bars, 200 μm). ( b ) qRT-PCR analysis of α-SMA mRNA expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica treatment (n = 3 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( c ) Western blot analysis of α-SMA expression in lung tissues from Fstl1 +/− and WT mice 21 days after saline or silica treatment. β-tubulin was used as a loading control.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Immunofluorescence, Expressing, Saline, Staining, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Fstl1 modulates myofibroblast differentiation via facilitating TGF-β1 signaling. ( a ) qRT-PCR analysis of TGF-β1 mRNA expression in lung tissues of Fstl1 +/− and WT mice at indicated time after saline or silica exposure (n = 4 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( b ) ELISA analysis of active form of TGF-β1 protein in lung tissues of Fstl1 +/− and WT mice at indicated time after saline or silica exposure (n = 4 per group; * P < 0.05 by one-way ANOVA followed by Student’s t test). ( c ) The levels of phosphorylation of Smad2/3 (p-Smad2/3), total Smad2/3 (T-Smad2/3), phosphorylation of Smad1/5 (p-Smad1/5) and total Smad1/5 (T-Smad1/5) in lung tissues of of Fstl1 +/− and WT mice 21 days after saline or silica exposure were determined by western blot analysis. β-tubulin was used as a loading control. ( d ) Primary lung fibroblasts from Fstl1 +/− and WT mice were treated with 5 ng/ml TGF-β1. Protein expressions of α-SMA in cell extracts and type I collagen (Col1) in medium 24 h after TGF-β1 treatment were determined by western blot analysis. β-tubulin was used as a loading control.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Saline, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: FSTL1-neutralizing antibody attenuates silica-induced lung inflammation and subsequent pulmonary fibrosis in mice. ( a ) In a FSTL1 blockage experiment, C57BL/6 mice were intraperitoneally injected with 22B6 mAb or IgG1 (n = 6 per group) every other day from 1 day after silica challenge till the mice were sacrificed on day 7 for inflammation analysis or day 21 for fibrosis analysis. ( b–f ) For inflammation analysis, ( b ) the number of total BALF cells was determined by hemocytometer (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( c–e ) The differential cell counts in BALF were determined according to standard morphologic criteria. ( c ) Macrophages (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( d ) PMNs. ( e ) Lymphocytes. ( f ) The level of cytokine IL-1β was detected by ELISA assay (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( g – i ) For fibrosis analysis, ( g ) hydroxyproline contents in lung tissues were measured (* P < 0.05, *** P < 0.001 by one-way ANOVA followed by Student’s t test). ( h ) Representative images of the H&E staining of lung sections are shown (Scale bars, 200 μm). ( i ) Lung fibrotic score analysis of the lung sections. The fibrotic area is presented as a percentage (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( j ) Interventional dosing regimen of lung fibrosis model. C57BL/6J mice were intraperitoneally injected with 22B6 mAb or IgG1 (n = 10 per group) at indicated time after silica exposure, and lungs were harvested on day 21. ( k ) Representative images of the H&E staining of lung sections are shown (Scale bars, 200 μm). ( l ) Lung fibrotic score analysis of the lung sections. The fibrotic area is presented as a percentage (* P < 0.05 by one-way ANOVA followed by Student’s t test). ( m ) Hydroxyproline contents in lung tissues (*** P < 0.001 by one-way ANOVA followed by Student’s t test).

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific Reports

Article Title: Follistatin like-1 aggravates silica-induced mouse lung injury

doi: 10.1038/s41598-017-00478-0

Figure Lengend Snippet: Clinical characteristics of the study groups.

Article Snippet: The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature.

Techniques:

Journal: BMC pulmonary medicine

Article Title: FSTL1 aggravates cigarette smoke-induced airway inflammation and airway remodeling by regulating autophagy.

doi: 10.1186/s12890-021-01409-6

Figure Lengend Snippet: Fig. 1 FSTL1 was highly expressed in COPD patients and correlated with autophagy. a FSTL1 level was significantly higher in the serum of COPD patients (n = 28) compared with control (n = 25) (ELISA). b Airway specimens of COPD showed inflammation infiltration, RBM fragmentation (black triangle), epithelial integrity loss, and cilia lodging (black arrow) (HE staining, ×400). c Positive areas of FSTL1 in airway specimens of COPD patients were significantly larger than those of control subjects (immunohistochemistry staining, ×400). d Positive areas of LC3B in airway specimens were significantly larger than control subjects (immunohistochemistry staining, ×400). e Quantification of IHC. Median of each group is presented. * difference between control and COPD group, p < 0.05; ***p < 0.001, t-test. Scale bar, 20 μm. Abbreviations: CON, control

Article Snippet: To detect the level of FSTL1, IL-8, TNF-α and LTB4, we tested the serum of patients and BALF of mice using enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO, MD, USA) according to the manufacturer’s instruction.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry

Journal: BMC pulmonary medicine

Article Title: FSTL1 aggravates cigarette smoke-induced airway inflammation and airway remodeling by regulating autophagy.

doi: 10.1186/s12890-021-01409-6

Figure Lengend Snippet: Fig. 5 The haplodeletion of FSTL1 attenuates autophagy in CS-exposed model. a The staining of FSTL1 and autophagic proteins LC3B, P62 in lung tissues of FSTL1± and FSTL1flox/+ mice with or without CS exposure (Immunohistochemistry, ×400. Scale bar, 20 μm.). b Autophagosome formation was inhibited in CS-FSTL1± mice. (Transmission electron microscopy. Scale bar,2 μm,500 nm). c The protein level of FSTL1, LC3B and P62 in each group (Western blotting). d Quantification of IHC and WB. Each group consisted of 6 mice, and the median of each group is presented. All the experiments were repeated independently at least 3 times. *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA, t-test. Abbreviations: CS, cigarette smoke

Article Snippet: To detect the level of FSTL1, IL-8, TNF-α and LTB4, we tested the serum of patients and BALF of mice using enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO, MD, USA) according to the manufacturer’s instruction.

Techniques: Staining, Immunohistochemistry, Transmission Assay, Electron Microscopy, Western Blot

Journal: BMC pulmonary medicine

Article Title: FSTL1 aggravates cigarette smoke-induced airway inflammation and airway remodeling by regulating autophagy.

doi: 10.1186/s12890-021-01409-6

Figure Lengend Snippet: Fig. 7 FSTL1 deficiency and autophagy inhibitor both alleviate lung function decline in CS-exposed mice. a After CS exposure, total lung capacity did not increase in the 3-MA pretreatment mice, and FSTL1± mice showed a similar protective effect as 3-MA versus FSTL1flox/+ mice. b After CS exposure, lung compliance did not increase sharply in the 3-MA pretreatment mice, and FSTL1± mice showed a similar protective effect as 3-MA versus FSTL1flox/+ mice. Each group consisted of 6 mice, and the median of each group is presented. All the experiments were repeated independently at least 3 times. *difference between CON-WT and CS-WT, FSTL1flox/+ and CS-FSTL1flox/+, p < 0.05; **p < 0.01; #difference between CS-WT and CS + 3MA-WT, CS-FSTL1± and CS-FSTL1flox/+, p < 0.05; one-way ANOVA. Abbreviations: CS, cigarette smoke. 3-MA, 3-methyladenine. CON, control. WT, wild type

Article Snippet: To detect the level of FSTL1, IL-8, TNF-α and LTB4, we tested the serum of patients and BALF of mice using enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO, MD, USA) according to the manufacturer’s instruction.

Techniques: Control

Journal: BMC pulmonary medicine

Article Title: FSTL1 aggravates cigarette smoke-induced airway inflammation and airway remodeling by regulating autophagy.

doi: 10.1186/s12890-021-01409-6

Figure Lengend Snippet: Fig. 6 FSTL1 deficiency abates airway remodeling and airway inflammation. a Positive areas of collagen I and α-SMA in lung tissues of FSTL1± and FSTL1flox/+ mice with or without CS exposure (immunohistochemistry, ×400). b The protein level of collagen I and α-SMA in each group (western blotting). c: Quantification of IHC and WB. d Levels of TNF-α, IL-8 and LTB4 increased significantly in BALF of CS-FSTL1flox/+ mice than CS-FSTL1± mice (ELISA). Each group consisted of 6 mice, and the median of each group is presented. All the experiments were repeated independently at least 3 times. *p < 0.05; **p < 0.01; one-way ANOVA. Scale bar, 20 μm. Abbreviations: CS, cigarette smoke. BALF, bronchoalveolar lavage fluid

Article Snippet: To detect the level of FSTL1, IL-8, TNF-α and LTB4, we tested the serum of patients and BALF of mice using enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO, MD, USA) according to the manufacturer’s instruction.

Techniques: Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: ZFAS1 knockdown inhibits fibroblast-like synoviocyte proliferation, migration, invasion and inflammation, and promotes apoptosis via miR-3926/FSTL1 in rheumatoid arthritis

doi: 10.3892/etm.2021.10346

Figure Lengend Snippet: miR-3926 directly targets FSTL1 and negatively regulates its expression in RA-FLSs. (A) Potential binding sites between miR-3926 and FSTL1 were predicted via TargetScan. (B) Dual-luciferase reporter assay was performed to determine luciferase activity in RA-FLSs co-transfected with FSTL1 3'UTR WT or FSTL1 3'UTR MUT and miR-3926 or miR-NC. (C) mRNA and (D) protein expression levels of FSTL1 in RA synovial and non-arthritic control tissue were analyzed via RT-qPCR and western blotting, respectively. (E) mRNA and (F) protein expression levels of FSTL1 in RA-FLSs and N-FLSs were assessed via RT-qPCR and western blotting, respectively. (G) Pearson's correlation analysis was utilized to analyze the correlation between miR-3926 and FSTL1 in RA synovial tissue. RA-FLSs were transfected with miR-NC, miR-3926, anti-miR-NC or anti-miR-3926 and the (H) mRNA and (I) protein expression levels of FSTL1 were determined via RT-qPCR and western blotting assay, respectively. *P<0.05 vs. miR-NC. miR, microRNA; FSTL1, follistatin-like protein 1; UTR, untranslated region; WT, wild type; MUT, mutant; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; RA, rheumatoid arthritis; FLSs, fibroblast-like synoviocytes; N, normal.

Article Snippet: Subsequently, the membranes were blocked in 5% skimmed milk for 2 h at room temperature and incubated overnight at 4˚C with primary antibodies (all BIOSS) against cleaved caspase-3 (C-caspase-3; cat. no. bs-0081R; 1:2,000), interleukin (IL)-6 (cat. no. bs-4539R; 1:2,000), IL-1β (cat. no. bs-20448R; 1:2,000), tumor necrosis factor (TNF)-α (cat. no. bs-10802R; 1:2,000), FSTL1 (cat. no. bs-6050R; 1:2,000) and GAPDH (cat. no. bs-0755R; 1:5,000), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (cat. no. bs-40296G-HRP; 1:10,000; BIOSS) for 1 h at room temperature.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Mutagenesis, Negative Control, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: ZFAS1 knockdown inhibits fibroblast-like synoviocyte proliferation, migration, invasion and inflammation, and promotes apoptosis via miR-3926/FSTL1 in rheumatoid arthritis

doi: 10.3892/etm.2021.10346

Figure Lengend Snippet: Inhibition of miR-3926 restores the inhibitory effect of FSTL1 knockdown on RA-FLSs development. RA-FLSs were transfected with si-NC, si-FSTL1, si-FSTL1 + anti-miR-NC or si-FSTL1 + anti-miR-3926. (A) mRNA and (B) protein expression levels of FSTL1 in RA-FLSs were determined via reverse transcription-quantitative PCR and western blotting, respectively. (C) Cell proliferation in RA-FLSs was assessed via MTT assay. (D) Cell apoptosis in RA-FLSs was evaluated via flow cytometric analysis. (E) Expression levels of C-caspase-3 in RA-FLSs were determined via western blotting. Cell (F) migration and (G) invasion of RA-FLSs were determined by Transwell assay (magnification, x100). (H) Expression levels of IL-6, IL-1β and TNF-α in RA-FLSs were assessed via western blotting. *P<0.05. miR, microRNA; FSTL1, follistatin-like protein 1; RA-FLSs, rheumatoid arthritis-fibroblast-like synoviocytes; si, small interfering RNA; NC, negative control; C-caspase-3, cleaved-caspase-3; IL, interleukin; TNF-α, tumor necrosis factor-α.

Article Snippet: Subsequently, the membranes were blocked in 5% skimmed milk for 2 h at room temperature and incubated overnight at 4˚C with primary antibodies (all BIOSS) against cleaved caspase-3 (C-caspase-3; cat. no. bs-0081R; 1:2,000), interleukin (IL)-6 (cat. no. bs-4539R; 1:2,000), IL-1β (cat. no. bs-20448R; 1:2,000), tumor necrosis factor (TNF)-α (cat. no. bs-10802R; 1:2,000), FSTL1 (cat. no. bs-6050R; 1:2,000) and GAPDH (cat. no. bs-0755R; 1:5,000), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (cat. no. bs-40296G-HRP; 1:10,000; BIOSS) for 1 h at room temperature.

Techniques: Inhibition, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Migration, Transwell Assay, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: ZFAS1 knockdown inhibits fibroblast-like synoviocyte proliferation, migration, invasion and inflammation, and promotes apoptosis via miR-3926/FSTL1 in rheumatoid arthritis

doi: 10.3892/etm.2021.10346

Figure Lengend Snippet: ZFAS1 positively modulates FSTL1 expression via miR-3926 in RA-FLSs. (A) Correlation between ZFAS1 and FSTL1 in RA synovial tissue was analyzed via Pearson's correlation analysis. (B) mRNA and (C) protein expression levels of FSTL1 in RA-FLSs transfected with si-NC, si-ZFAS1, si-ZFAS1 + anti-miR-NC or si-ZFAS1 + anti-miR-3926 were assessed via reverse transcription quantitative PCR and western blotting, respectively. *P<0.05. ZFAS1, zinc finger antisense 1; FSTL1, follistatin-like protein 1; miR, microRNA; RA-FLSs, rheumatoid arthritis-fibroblast-like synoviocytes; si, small interfering RNA; NC, negative control.

Article Snippet: Subsequently, the membranes were blocked in 5% skimmed milk for 2 h at room temperature and incubated overnight at 4˚C with primary antibodies (all BIOSS) against cleaved caspase-3 (C-caspase-3; cat. no. bs-0081R; 1:2,000), interleukin (IL)-6 (cat. no. bs-4539R; 1:2,000), IL-1β (cat. no. bs-20448R; 1:2,000), tumor necrosis factor (TNF)-α (cat. no. bs-10802R; 1:2,000), FSTL1 (cat. no. bs-6050R; 1:2,000) and GAPDH (cat. no. bs-0755R; 1:5,000), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (cat. no. bs-40296G-HRP; 1:10,000; BIOSS) for 1 h at room temperature.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Negative Control