Journal: The Journal of Craniofacial Surgery

Article Title: FSTL1 Can Be a Promising Target in TMJ Osteoarthritis via Regulating Chondrocyte Mitophagy and Apoptosis

doi: 10.1097/SCS.0000000000010906

Figure Lengend Snippet: (A) Typical CBCT scan of TMD patients without OA. The red arrow showed the relatively normal bone margins. (B) Typical CBCT scan of TMD patients with OA. The red arrow showed the bone resorption in condyle. (C) The ELISA results of FSTL1 in TMJ auricular synovium fluid. (D) The ROC curve of the diagnosis value FSTL1 concentration in TMJ OA progress. CBCT indicates cone beam computed tomography; FSTL1, follistatin-like protein 1; OA, osteoarthritis; ROC, receiver operating characteristic; TMD, temporomandibular joint disorder; TMJ, temporomandibular joint.

Article Snippet: The used antibodies were as followed, FSTL1 (1:200, Proteintech), ACAN (1:200, Proteintech), COL2A1(1:200, Millipore), and MMP13 (1:200, Abclonal).

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Concentration Assay, Computed Tomography

Journal: The Journal of Craniofacial Surgery

Article Title: FSTL1 Can Be a Promising Target in TMJ Osteoarthritis via Regulating Chondrocyte Mitophagy and Apoptosis

doi: 10.1097/SCS.0000000000010906

Figure Lengend Snippet: (A) The Rt-qPCR results of FSTL1 with the targeting plasmid transfection. (B) The western blot results of FSTL1 with the targeting plasmid transfection. (C) The Rt-qPCR results of the mRNA fold change of related typical markers to chondrocyte synthesis and catabolism as well as under the inflammatory stimulation. (D) The western blot results of changes of related typical markers to chondrocyte synthesis and catabolism as well as under the inflammatory stimulation. (E) The safranine O staining of chondrogenic cells with / without FSTL1 overexpression. FSTL1 indicates follistatin-like protein 1; Rt-qPCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: The used antibodies were as followed, FSTL1 (1:200, Proteintech), ACAN (1:200, Proteintech), COL2A1(1:200, Millipore), and MMP13 (1:200, Abclonal).

Techniques: Quantitative RT-PCR, Plasmid Preparation, Transfection, Western Blot, Staining, Over Expression, Reverse Transcription, Polymerase Chain Reaction

Journal: The Journal of Craniofacial Surgery

Article Title: FSTL1 Can Be a Promising Target in TMJ Osteoarthritis via Regulating Chondrocyte Mitophagy and Apoptosis

doi: 10.1097/SCS.0000000000010906

Figure Lengend Snippet: (A, B) The mitosox probe staining and the quantified analysis of the mitochondrial morphology and function with / without FSTL1 overexpression. (C, D) The western blot and the quantified analysis results of mitochondrial function with / without FSTL1 overexpression and inflammatory stimulation. (E, F) Flow cytometry analysis of the chondrocytes apoptosis rate with / without FSTL1 overexpression. (G, H) Flow cytometry analysis of the chondrocytes cell cycle with / without FSTL1 overexpression. FSTL1 indicates follistatin-like protein 1.

Article Snippet: The used antibodies were as followed, FSTL1 (1:200, Proteintech), ACAN (1:200, Proteintech), COL2A1(1:200, Millipore), and MMP13 (1:200, Abclonal).

Techniques: Staining, Over Expression, Western Blot, Flow Cytometry

Journal: The Journal of Craniofacial Surgery

Article Title: FSTL1 Can Be a Promising Target in TMJ Osteoarthritis via Regulating Chondrocyte Mitophagy and Apoptosis

doi: 10.1097/SCS.0000000000010906

Figure Lengend Snippet: (A) The Rt-qPCR results mRNA fold change with the targeted si-FSTL1. (B) The western blot results of FSTL1 with the targeting si-RNA and its impact of typical markers to chondrocyte synthesis and catabolism. (C, D) The mitosox probe and IF staining of mt-COⅡ and the quantified analysis of the mitochondrial morphology and function with / without downregulation of FSTL1 under inflammatory stimulation. (E, F) The western blot and the quantified analysis results of mitochondrial function with / without downregulation of FSTL1 and inflammatory stimulation. (G, H) Flow cytometry analysis of the chondrocytes apoptosis rate with / without FSTL1 downregulation. FSTL1 indicates follistatin-like protein 1; Rt-qPCR, quantitative reverse transcription polymerase chain reaction

Article Snippet: The used antibodies were as followed, FSTL1 (1:200, Proteintech), ACAN (1:200, Proteintech), COL2A1(1:200, Millipore), and MMP13 (1:200, Abclonal).

Techniques: Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry, Reverse Transcription, Polymerase Chain Reaction

Journal: The Journal of Craniofacial Surgery

Article Title: FSTL1 Can Be a Promising Target in TMJ Osteoarthritis via Regulating Chondrocyte Mitophagy and Apoptosis

doi: 10.1097/SCS.0000000000010906

Figure Lengend Snippet: (A, B) HE and safranin O/Fast green staining of the cartilage in the sham group and UAC-induced OA rats with si-NC or si-FSTL1 injected group. Scale bar=100 μm. (C) IHC staining of the cartilage in the sham group and UAC-induced OA rats with si-NC or si-FSTL1 injected group. Scale bar=100 μm in the larger field of view and 50 μm in the smaller field of view. (D) The gross OARSI score of the cartilage condition in the sham group and UAC-induced OA rats with si-NC or si-FSTL1 injected group. (E) The statistical analysis of the cartilage IHC staining in the sham group and UAC-induced OA rats with si-NC or si-FSTL1 injected group. FSTL1 indicates follistatin-like protein 1; HE, hematoxylin-eosin; IHC, immunohistochemical; OA, osteoarthritis; UAC, unilateral anterior crossbite.

Article Snippet: The used antibodies were as followed, FSTL1 (1:200, Proteintech), ACAN (1:200, Proteintech), COL2A1(1:200, Millipore), and MMP13 (1:200, Abclonal).

Techniques: Staining, Injection, Immunohistochemistry, Immunohistochemical staining

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Demographics of colorectal cancer patients and the statistical association with FSTL1/DIP2A expressions in the tumor tissues

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques:

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Apoptotic T cells increase in the mice with FSTL1+ colorectal cancer. A. Establishment of FSTL1 transfectants (TR1-3) using murine colorectal cancer (CRC) MC38 cells. Photos show morphological changes (round types to spindle types) after fstl1 transduction (scale = 1,000 µm). FSTL1 in the cultured supernatants was measured by ELISA, and cellular functions were tested (n = 3). B. Retardation of the TR tumor growth after subcutaneous implantation in mice (n = 5). C. Increase of PD1+ annexin V+ apoptotic T cells within the TR tumors (day 14; n = 5). D. Increase of PD1+ annexin V+ apoptotic T cells in spleen of the TR-implanted mice (day 14; n = 5). E. Expansion of a DIP2A+LAG3+ subset in the CD11b+ cells of the TR-implanted mice (day 14; n = 5). Graphs show means ± SDs except scatter plots (open circles, individual data; closed circles, means). *P < 0.01, **P < 0.05 versus mock control. Representative data of three independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: The T-cell apoptosis is caused by the FSTL1+ tumor-induced CD11b+DIP2A+LAG3+ cells. (A) FSTL1 upregulates LAG3 expression in CD11b+ cells. Splenic CD11b+ cells obtained from naive mice were stimulated with FSTL1 in the presence or absence of anti-FSTL1 mAb, anti-LAG3 mAb, or isotype control for 3 days, and were analyzed by flow cytometry. (B, C) The TR-stimulated CD11b+ cells suppress CD8+ T-cell proliferation partly via LAG3. Splenic CD11b+ cells obtained from tumor-implanted mice (day 14) were cocultured (2:1) with CD8+ T cells (2:1) in the presence of anti-CD3 mAb and/or anti-LAG3 mAb for 5 days (n = 3). CFSE-labeled CD8+ T cells were used for tracking cell division (B). The cocultured CD8+ T cells were analysed for expression of immune checkpoint molecules by flow cytometry (C). (D) In vivo T-cell exclusion caused by the TR-stimulated CD11b+ cells partly via the LAG3. MC38 cells were s.c. coinjected (1:1) with the CD11b+ cells in mice, and 4 days later, the mice were treated with anti-LAG3 mAb or isotype control at 10 mg/kg (n = 5). Twenty days after coinjection, tumors and spleens were harvested for flow cytometric analysis. *P < 0.01, **P < 0.05. Graphs show means ± SDs. Representative data of three independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Expressing, Flow Cytometry, Labeling, In Vivo

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Blocking LAG3 interferes the FSTL1-induced T-cell suppression in human system. A. Establishment of FSTL1 transfectants (TR1-3) using human CRC HCT116 cells. Photos show morphological changes (round types to spindle types) and DIP2A enhancement after fstl1 transduction (scale = 100 µm). FSTL1 in the cultured supernatants was measured by ELISA, and cellular functions were tested (n = 3). *P < 0.01 versus mock control. B. The TR-induced CD11b+ cells suppress T-cell proliferation via LAG3. CD11b+ were sorted from human PBMCs, and were stimulated with tumor supernatant fluids for 5 days followed by flow cytometric analysis for LAG3 and DIP2A expressions. CD3+ T cells were cocultured (2:1) with the stimulated CD11b+ cells in the presence of anti-CD3 mAb and/or anti-LAG3 mAb for 5 days (n = 3). **P < 0.05. Graphs show means ± SDs. Representative data of two independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Blocking Assay, Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Blocking FSTL1 and LAG3 synergizes in treatment of mouse CRC models. (A-C) Blocking LAG3 successfully elicits anti-tumor effects by suppressing CD8+ T-cell apoptosis in the TR-implanted mice. Mice were s.c. implanted with tumor cells, and were i.p. injected with anti-PD1 mAb, anti-LAG3 mAb, or rat IgG as a control (10 mg/kg) on days 4 and 7 after tumor implantation (n = 5). Tumor growth was measured (A), and tumors and spleen were harvested for flow cytometric analysis (B) and cytotoxic assay (E:T ratio = 20:1; C) on day 18. (D) Blocking LAG3 synergizes with blocking FSTL1 in the treatment of CRC metastasis models. Mice were both s.c. and intravenously (i.v.) implanted with FSTL1+ Colon26 cells, and were i.p. injected with anti-LAG3 mAb, anti-FSTL1 mAb, and/or rat IgG (5 mg/kg) on days 4 and 7 (n = 5). *P < 0.01, **P < 0.05 versus control group. Graphs show means ± SDs. Representative data of three independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Blocking Assay, Injection, Tumor Implantation

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: FSTL1 positivity in tumor tissues significantly correlates with poor prognosis of CRC patients. Tumor tissues obtained from CRC patients were immunohistochemically analyzed for FSTL1 and DIP2A expressions (n = 306). A. Representative photos (scale = 100 µm). B. Significant correlation between FSTL1 and DIP2A expressions. The molecular expression patterns were categorized into 4 levels according to the immunofluorescence intensity depicted as pixel counts: Level 0, no expression; Level 1, weak expression; Level 2, moderate expression; and Level 3, strong expression. C. FSTL1 positivity is reversely correlated with overall survival of the patients.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Expressing, Immunofluorescence

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: CD11b+DIP2A+LAG3+ cells are expanded in peripheral blood of metastatic CRC patients. PBMCs were obtained from healthy donors (n = 4) and patients with stage IV metastatic CRC (n = 11). A. FSTL1 concentration measured by ELISA (means ± SDs). B. Gating strategy and representative dot plot data in flow cytometric analysis. C. Significant correlation between CD11b+DIP2A+LAG3+ cell expansion and CD3+CD8+Ki67+GZMB+ T-cell reduction. Open circles, healthy donors. Closed circles, CRC patients. The P value in the scatter plot was analyzed by the nonparametric Spearman’s rank test.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay