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: Molecular Therapy. Nucleic Acids

Article Title: A Discovery of Clinically Approved Formula FBRP for Repositioning to Treat HCC by Inhibiting PI3K/AKT/NF-κB Activation

doi: 10.1016/j.omtn.2019.12.023

Figure Lengend Snippet: Illustration of PI3K-Akt-NF-κB Signaling, which Is Associated with the Repositioned Anti-HCC Efficacy of FBRP

Article Snippet: The detailed information on these antibodies is as follows: p-PI3K (catalog no. 4249, Cell Signaling Technology, MA, USA), p-AKT (catalog no. 4060, Cell Signaling Technology, MA, USA), IKκB (catalog no. 8943, Cell Signaling Technology, MA, USA), p-IKκB (catalog no. YP0637, ImmunoWay, TX, USA), NF-κB (p65) (catalog no. YP0191, ImmunoWay, TX, USA), p-NF-κB (p65) (catalog no. 3033, Cell Signaling Technology, MA, USA), CCND1 (catalog no. 2978, Cell Signaling Technology, MA, USA), CCNE1 (catalog no. 20808, Cell Signaling Technology, MA, USA), CDK2 (catalog no. 10122-1-AP, Proteintech, IL, USA), CDK4 (catalog no. 11026-1-AP, Proteintech, IL, USA), and GAPDH (catalog no. 2118, Cell Signaling Technology, MA, USA).

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: A Discovery of Clinically Approved Formula FBRP for Repositioning to Treat HCC by Inhibiting PI3K/AKT/NF-κB Activation

doi: 10.1016/j.omtn.2019.12.023

Figure Lengend Snippet: Effects of FBRP on Expression Levels of Candidate Targets Involved in PI3K/AKT/NF-κB Signaling in Liver Tissues of DEN-Induced HCC Rats (A) Western blots of the candidate targets of FBRP against HCC involved in PI3K/AKT/NF-κB signaling. (B–M) Relative protein expression levels of (B) p-PI3K, (C) p-AKT, (D) IKκB, (E) p-IKκB, (F) the ratio of p-IKκB to IKκB, (G) NF-κB (p65), (H) p-NF-κB (p65), (I) the ratio of NF-κB (p65) to p-NF-κB (p65), (J) CCND1, (K) CCNE1, (L) CDK2, and (M) CDK4 in different groups are shown. Data are expressed as the mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001, comparison with the normal group; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001, comparison with the DEN-12w group; *p < 0.05, **p < 0.01, ***p < 0.001, comparison with the DEN-15w group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, comparison with the DEN-18w group.

Article Snippet: The detailed information on these antibodies is as follows: p-PI3K (catalog no. 4249, Cell Signaling Technology, MA, USA), p-AKT (catalog no. 4060, Cell Signaling Technology, MA, USA), IKκB (catalog no. 8943, Cell Signaling Technology, MA, USA), p-IKκB (catalog no. YP0637, ImmunoWay, TX, USA), NF-κB (p65) (catalog no. YP0191, ImmunoWay, TX, USA), p-NF-κB (p65) (catalog no. 3033, Cell Signaling Technology, MA, USA), CCND1 (catalog no. 2978, Cell Signaling Technology, MA, USA), CCNE1 (catalog no. 20808, Cell Signaling Technology, MA, USA), CDK2 (catalog no. 10122-1-AP, Proteintech, IL, USA), CDK4 (catalog no. 11026-1-AP, Proteintech, IL, USA), and GAPDH (catalog no. 2118, Cell Signaling Technology, MA, USA).

Techniques: Expressing, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: A Discovery of Clinically Approved Formula FBRP for Repositioning to Treat HCC by Inhibiting PI3K/AKT/NF-κB Activation

doi: 10.1016/j.omtn.2019.12.023

Figure Lengend Snippet: The Immunohistochemistry Analysis of the Effects of FBRP on the Subcellular Localization and Expression Levels of the Downstream Factors in PI3K/AKT/NF-κB Signaling in Liver Tissues of DEN-Induced HCC Rats and Their Quantification Results (A–E) Effects of FBRP on the expression of (A) CCND1, (B) CCNE1, (C) PCNA, (D) CDK2, and (E) CDK4 in the liver tissues of DEN-induced HCC rats examined by immunohistochemistry. Original magnification, ×400. Data are expressed as the mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001, comparison with the normal group; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001, comparison with the normal+FBRP group; *p < 0.05, **p < 0.01, ***p < 0.001, comparison with the DEN group.

Article Snippet: The detailed information on these antibodies is as follows: p-PI3K (catalog no. 4249, Cell Signaling Technology, MA, USA), p-AKT (catalog no. 4060, Cell Signaling Technology, MA, USA), IKκB (catalog no. 8943, Cell Signaling Technology, MA, USA), p-IKκB (catalog no. YP0637, ImmunoWay, TX, USA), NF-κB (p65) (catalog no. YP0191, ImmunoWay, TX, USA), p-NF-κB (p65) (catalog no. 3033, Cell Signaling Technology, MA, USA), CCND1 (catalog no. 2978, Cell Signaling Technology, MA, USA), CCNE1 (catalog no. 20808, Cell Signaling Technology, MA, USA), CDK2 (catalog no. 10122-1-AP, Proteintech, IL, USA), CDK4 (catalog no. 11026-1-AP, Proteintech, IL, USA), and GAPDH (catalog no. 2118, Cell Signaling Technology, MA, USA).

Techniques: Immunohistochemistry, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: A Discovery of Clinically Approved Formula FBRP for Repositioning to Treat HCC by Inhibiting PI3K/AKT/NF-κB Activation

doi: 10.1016/j.omtn.2019.12.023

Figure Lengend Snippet: Effects of FBRP on Expression Levels of Candidate Targets Involved in PI3K/AKT/NF-κB Signaling in the HCC Cell Line Huh7 (A) Western blots of the candidate targets of FBRP against HCC involved in PI3K/AKT/NF-κB signaling in different groups. (B–M) Relative protein expression levels of (B) p-PI3K, (C) p-AKT, (D) IKκB, (E) p-IKκB, (F) the ratio of p-IKκB to IKκB, (G) NF-κB (p65), (H) p-NF-κB (p65), (I) the ratio of NF-κB (p65) to p-NF-κB (p65), (J) CCND1, (K) CCNE1, (L) CDK2, and (M) CDK4 in different groups are shown. Data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, comparison with the Huh7 group; # p < 0.05, ## p < 0.01, ### p < 0.001, comparison with the Huh7-FBRP treatment group; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001, comparison with the Huh7-LY294002 group.

Article Snippet: The detailed information on these antibodies is as follows: p-PI3K (catalog no. 4249, Cell Signaling Technology, MA, USA), p-AKT (catalog no. 4060, Cell Signaling Technology, MA, USA), IKκB (catalog no. 8943, Cell Signaling Technology, MA, USA), p-IKκB (catalog no. YP0637, ImmunoWay, TX, USA), NF-κB (p65) (catalog no. YP0191, ImmunoWay, TX, USA), p-NF-κB (p65) (catalog no. 3033, Cell Signaling Technology, MA, USA), CCND1 (catalog no. 2978, Cell Signaling Technology, MA, USA), CCNE1 (catalog no. 20808, Cell Signaling Technology, MA, USA), CDK2 (catalog no. 10122-1-AP, Proteintech, IL, USA), CDK4 (catalog no. 11026-1-AP, Proteintech, IL, USA), and GAPDH (catalog no. 2118, Cell Signaling Technology, MA, USA).

Techniques: Expressing, Western Blot