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: medRxiv

Article Title: IRAK2 deficiency causes a new immune dysregulation disorder

doi: 10.1101/2024.10.21.24315796

Figure Lengend Snippet: (A) Co-immunoprecipitation of whole cell lysates from HEK293T cells transiently expressing Myc-tagged IRAK2-WT or IRAK2-Δex2 and equal levels of IRAK4-Flag or TRAF6-Flag using anti-Flag antibody (IP: Flag). The experiment was repeated three times. (B) Immunoblot showing deficiency of NF- κ B and MAPK signaling in the presence of IRAK2-Δex2 mutation compared with IRAK2-WT, using indicated antibodies in the stable HEK293T-TLR4 cell line. The experiment was repeated three times. (C) NF- κ B signaling was detected by luciferase assay in HEK293T- IRAK2 −/− cells overexpressing either IRAK2-WT or IRAK2-Δex2. Cells were co-transfected with NF- κ B-luciferase reporter and renilla plasmids (n=3). Bars represent the mean ± SD. An unpaired Student’s t -test was performed between the IRAK2-WT and IRAK2-Δex2 groups, ** P < 0.01. The experiment was repeated three times. (D) RNA sequencing analysis of NF- κ B pathway in PBMCs treated with 1 μg/ml LPS, 1 μg/ml R848, respectively, or left untreated for 12 hours. (E) PBMCs from P1, P2, and six unaffected controls were treated with 1 μg/ml LPS, 1 μg/ml R848, respectively, or left untreated for 12 hours. Supernatants were collected for CBA. Bars represent the mean ± SD. (F) RNA sequencing analysis of NF- κ B pathway in BMDMs treated with 100 ng/ml LPS or left untreated for 8 hours (n=3). (G) Quantitative PCR (qPCR) analysis of the expression of the genes related to NF- κ B signaling in BMDMs treated with 100 ng/ml LPS, 25 ng/ml R848, or 50 ng/ml FSL-1, respectively, or left untreated for 8 hours (n=5). Bars represent the mean ± SEM. Unpaired Student’s t -tests were performed between the Irak2 WT/WT and Irak2 Δ ex2/ Δ ex2 groups, * P < 0.05, ** P < 0.01, *** P < 0.001. UNS, unstimulated; ns, not significant.

Article Snippet: BMDMs were stimulated with 100 ng/ml LPS, 25 ng/ml R848 (HY-13740, MCE), and 50 ng/ml FSL-1 (HY-P2036, MCE), respectively for 8 hours prior to RNA extraction.

Techniques: Immunoprecipitation, Expressing, Western Blot, Mutagenesis, Luciferase, Transfection, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: medRxiv

Article Title: IRAK2 deficiency causes a new immune dysregulation disorder

doi: 10.1101/2024.10.21.24315796

Figure Lengend Snippet: (A) The gene set enrichment analysis (GSEA) of RNA sequencing data for PBMCs. NES, normalized enrichment score. (B) Quantification of the 28-gene IFN score of RNA sequencing data of total RNA extracted from PBMCs. Data are presented as the mean ± SD. Significance assessed by unpaired Student’s t -test, *** P < 0.001, ** P < 0.01. (C) Uniform Manifold Approximation and Projection (UMAP) visualization and marker-based annotation of 13 cell subtypes from P1, P2, and three unaffected controls. ‘Macro-like’ cell subset is circled in red. NK, natural killer cells; Treg, regulatory T cells; pDC, plasmacytoid dendritic cells; mDC, myeloid dendritic cells; Macro-like: macrophage-like. (D) Identification of differentially expressed genes between patients (P1 and P2) and three unaffected controls across different cell types. Each point denotes a gene with an adjusted p-value (method= “BH”) less than 0.05. The dashed line represents a Log2FC value of 1, marking the number of differentially expressed genes exceeding this threshold across cell clusters. (E) The bubble graph shows the expression of marker genes for annotating monocytes and ‘Macro-like’ cell subset by scRNA-seq. The size of the bubble indicates the proportion of cells expressing the gene, and the color scale indicates the average expression level. (F) The box plot represents the type I interferon response scores calculated by scRNA-seq for ‘Macro-like’ cell subset. Statistical analysis was performed using Wilcoxon test (*** P < 0.001). (G) The violin plots show the upregulation of IFN-stimulated genes in the ‘Macro-like’ cell subset of P1 and P2 compared to three unaffected controls. (H) The phosphorylation level of STAT1 of CD16+ monocytes within PBMCs from P3 and three unaffected controls, as determined by flow cytometry analysis. (I) qPCR analysis of Ifnβ expression in BMDMs treated with 100 ng/ml LPS, 25 ng/ml R848, or 50 ng/ml FSL-1, respectively, or left untreated for 8 hours (n=5). Bars represent the mean ± SEM. An unpaired Student’s t -test was conducted between Irak2 WT/WT and Irak2 Δ ex2/ Δ ex2 groups, *** P < 0.001.

Article Snippet: BMDMs were stimulated with 100 ng/ml LPS, 25 ng/ml R848 (HY-13740, MCE), and 50 ng/ml FSL-1 (HY-P2036, MCE), respectively for 8 hours prior to RNA extraction.

Techniques: RNA Sequencing, Marker, Expressing, Phospho-proteomics, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis *

doi: 10.1074/jbc.M901528200

Figure Lengend Snippet: The inflammatory stimuli of BMFE are lipoproteins that primarily signal via TLR2/6. A , HEK-TLR2 cells were transfected with plasmids encoding small interfering (psi) RNA specific for TLR1 or TLR6 before being stimulated with BMFE or control stimuli (doses stated are in micrograms/ml). Accumulations of IL-8 secreted by HEK-psiTLR1 or -psiTLR6 triplicate cultures 20 h post-stimulation are plotted as mean (±1 S.E.) percentages of corresponding HEK-TLR2 responses (mean ± 1 S.E. max IL-8 concentrations as follows: TNFα = 7859 ± 98 pg/ml, PAM 3 CSK = 9807 ± 175 pg/ml, FSL-1 = 2001 ± 345 pg/ml, and BMFE = 9495 ± 137 pg/ml). Significant differences compared with HEK-TLR2 responses are indicated: ***, p < 0.001; **, p < 0.01. B , peritoneal macrophages from WT, TLR1 −/− , TLR6 −/− were stimulated with BMFE in triplicate (doses stated are micrograms/ml), and production of TNFα after 20 h is plotted as mean ± 1 S.E. percentages of WT response to 400 μg/ml BMFE (mean ± 1S.E. max TNFα concentration = 188.4 ± 10.36 pg/ml). Significant differences compared with WT are indicated ***, p < 0.001; **, p < 0.01. C , triplicate HEK-TLR2 cultures were stimulated with BMFE or control stimuli (doses stated are micrograms/ml) before or following Cleanascite TM or BindPro TM treatment. Data plotted are mean IL-8 ± 1S.E. All data are representative of three independent experiments.

Article Snippet: Ultra-pure LPS, PAM 3 CSK 4 , FSL-1 (Autogen BioClear), and rTNFα (R&D Systems) were used at the doses stated.

Techniques: Transfection, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis *

doi: 10.1074/jbc.M901528200

Figure Lengend Snippet: Synthetic diacyl-lipopeptide analogue of wBmPAL (Diacyl WoLP) replicates BMFE-TLR2/6-specific activation of inflammation. A , HEK-TLR2 cells were transfected with plasmids encoding small interfering (psi) RNA specific for TLR1 or TLR6 before being stimulated with Diacyl WoLP or Triacyl WoLP (doses stated are in micrograms/ml). Accumulations of IL-8 secreted by HEK-psiTLR1 or -psiTLR6 triplicate cultures 20 h post-stimulation are plotted as mean ± 1 S.E. percentages of corresponding HEK-TLR2 responses (mean ± 1S.E. max IL-8 concentrations are as follows: PAM 3 CSK = 9807 ± 175 pg/ml, FSL-1 = 2001 ± 345 pg/ml, Diacyl WoLP = 8195 ± 199 pg/ml, Triacyl WoLP 571 ± 27 pg/ml, and BMFE = 9495 ± 137 pg/ml). Significant differences compared with HEK-TLR2 responses are indicated: ***, p < 0.001; **, p < 0.01; and *, p < 0.05. B , peritoneal macrophages from WT, TLR1 −/− , and TLR6 −/− were stimulated with Diacyl WoLP or Triacyl WoLP and control TLR1/6 ligands PAM 3 CSK 4 and FSL-1 (doses stated are in nanograms/ml) in triplicate, and production of TNFα after 20 h is plotted as mean ± 1S.E. All data are representative of three independent experiments.

Article Snippet: Ultra-pure LPS, PAM 3 CSK 4 , FSL-1 (Autogen BioClear), and rTNFα (R&D Systems) were used at the doses stated.

Techniques: Activation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis *

doi: 10.1074/jbc.M901528200

Figure Lengend Snippet: DC maturation and activation by Wolbachia and Diacyl WoLP requires MyD88, TLR2, and TLR6 but not TLR4. A , increase in CD80 or CD86 surface molecules following 20-h exposure to LPS, FSL-1, Diacyl WoLP, or BMFE in DC derived from WT, MyD88 −/− , TLR2 −/− , TLR4 −/− ,or TLR6 −/− mice. Doses stated are in micrograms/ml. Bars represent mean fold increase in MFI ± S.E. compared with unstimulated cells of triplicate labeling reactions. B , stimulation of TNFα, IL-12/IL-23p40, and IL-12p70 by LPS, FSL-1, Diacyl WoLP, or BMFE (doses stated are in micrograms/ml) from DC derived from WT, MyD88 −/− , TLR2 −/− , TLR4 −/− , or TLR6 −/− mice. Bars are mean ± 1S.E. cytokine production from triplicate cultures. C , Diacyl WoLP mediates an expansion of mature CD11c + DC in vivo . Increases in CD86 surface expression on CD11c + splenocytes 6 h following intraperitoneal inoculation with 50 μg of Diacyl WoLP were compared with sham inoculated WT mice. Numbers are percentages of splenocytes in the upper left and right quadrants. D , significant differences in CD80 and CD86 MFI on CD11c + splenocytes derived from WT mice were compared with TLR2 −/− -deficient mice 6 h following inoculation with 50 μg of Diacyl WoLP intraperitoneally. Bars are mean MFI from groups of three mice. E , significant differences in levels of IL-12/IL-23 p40 measured in spleen extracts from WT mice compared with TLR2 −/− -deficient mice 6 h following inoculation with 50 μg of Diacyl WoLP intraperitoneally. Bars are mean cytokine levels from groups of three mice. Significant reductions compared with WT are indicated: ***, p < 0.001; **, p < 0.01; and *, p < 0.05. All data are representative of two independent experiments.

Article Snippet: Ultra-pure LPS, PAM 3 CSK 4 , FSL-1 (Autogen BioClear), and rTNFα (R&D Systems) were used at the doses stated.

Techniques: Activation Assay, Derivative Assay, Labeling, In Vivo, Expressing

Journal: Respiratory Research

Article Title: Differential regulation of CCL-11/eotaxin-1 and CXCL-8/IL-8 by Gram-positive and Gram-negative bacteria in human airway smooth muscle cells

doi: 10.1186/1465-9921-9-30

Figure Lengend Snippet: Effect of bacteria and associated PAMPs on CXCL-8 release from human ASMCs over 24 h. Panel A, human ASMCs treated with Gram-negative bacteria , LPS or FK565. Panel B, cells treated with Gram-positive bacteria, Pam 3 CSK4, FSL-1, MDPLys18. Data represent mean ± SEM for triplicate of three different donors. * p < 0.05; **p < 0.01; ***p < 0.001 compared with un-stimulated control (medium only).

Article Snippet: The Pam 3 CSK4, LPS and FSL-1 were purchased from Axxora (UK) Ltd (Nottingham, U.K).

Techniques: