Journal: bioRxiv

Article Title: CD36: Hemin interaction axis to control immune responses and cytokine secretion from macrophages involving Lyn kinase

doi: 10.1101/2021.06.06.447270

Figure Lengend Snippet: Macrophages were stimulated with various concentrations of hemin (in µM) and analysed for cytokine secretion. (A) The macrophages either treated with hemin (25 or 200 µM) or remain untreated were allowed to secrete the cytokines overnight and the next day the cell culture supernatant were collected. The culture supernatants were assessed for the presence of cytokines using muse cytokine array panel A kit. The upper blot (untreated) shows the presence of various constitutively expressing cytokines along with control spots and a negative control spot. The hemin treated cytokine arrays (middle (25 µM) and bottom blot (200 µM) showing upregulated cytokines such as CCL1, IL-16, CXCL1, RANTES, MCP-1 and TNF-α. (B) The cytokine dot intensity was semi quantified and normalized by control spots provided in the array. The cytokine levels plotted as normalized pixel density (folds change) calculated using ImageJ application (NIH, USA). The cytokine such as TNF-α, RANTES and MCP1, G-CSF, CCL1 and sICAM1 showing upregulated. (C) Macrophages were treated with different concentration of hemin (0-200µM) for 1hrs at 37 0 C in serum free media. Cells were washed with PBS and allowed to secrete TNF-α for 12hrs in serum free media. TNF-α secretion from the cells were measured using ELISA as per manufacturer’s instruction and expressed as pg ±SD. n=3 (D) Macrophages either remained untreated or treated with hemin (75 µM) for 1 hrs at 37 0 C in serum free media. Cells were washed with PBS and stimulated with LPS (1µg/ml) for 1hrs, washed to remove LPS and allowed to secrete TNF-α for 12hrs in serum free media. TNF-α secretion from the cells was measured using ELISA as per manufacturer’s instruction and expressed as fold change. (E) Macrophages either remained untreated or treated with hemin (75 µM) for 1hrs in serum free media. Cells were washed with PBS and stimulated with LPS (1 µg/ml) for 1hrs, washed to remove LPS and allowed to secrete TNF-α for different time period (1-16 hrs) in serum free media. At each time point, culture supernatant was collected and TNF-α level was measured using ELISA as per manufacturer’s instruction and expressed as fold change.

Article Snippet: The cytokine ELISA kits for TNF-α (cat#. KRBA10602-5), MCP1 (cat#.KRB10134-5) and RANTES (cat#.KB1102) were purchased from Krishgen Biosystems, Mumbai, India and CCL1 (cat#.KTL11769) from (Kreative Technolabs, Los Angeles, CA, USA)

Techniques: Cell Culture, Expressing, Negative Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: CD36: Hemin interaction axis to control immune responses and cytokine secretion from macrophages involving Lyn kinase

doi: 10.1101/2021.06.06.447270

Figure Lengend Snippet: (A) Hemin interacts with CD36 at cellular levels. The MG63 cells transfected with either CD36 or mutants (R292A, D372A and Q382A) and carried out migration assay in presence or absence of hemin. The number of cells migrated in CD36 transfected MG63 in presence of hemin is significant and indicates the hemin interacts with the cell membrane bound scavenger receptor CD36. (n=3). (B) The images captured after cell migration in CD36 or mutants transfected cells in presence or absence of hemin. The upper panel shows absence of hemin and the lower panel for cells migrated in presence of hemin. There is no clear distinction was observed in mutants or mock transfected cells either in presence or absence of hemin. (C) Phagocytic activity of CD36 or mutants transfected MG63 cells. The cell after transfection was either treated with hemin or remain untreated and performed the phagocytosis assay as described in materials and methods section. The results clearly indicates at 25 μM hemin the phagocytic index increased to 30% compared to untreated and CD36 transfected cells. There is no significant change was observed in mutants transfected 25 μM hemin treated cells. (D) Bactericidal activity of CD36 or mutant transfected MG63 cells. There is no considerable changes were observed in bactericidal activity either hemin treated or untreated transfected cells. The MG63 cells transfected with CD36 or mutants and treated with hemin (25 or 200 μM). The cytokines TNF-α (E) , RANTES/CCL5 (F) , MCP-1 (G) , and CCL-1 (H) were quantified and expressed in folds change. All four cytokines were upregulated to several folds in 25 μM hemin treatment whereas two cytokines RANTES and CCL1 were reduced in 200 μM hemin treated condition.

Article Snippet: The cytokine ELISA kits for TNF-α (cat#. KRBA10602-5), MCP1 (cat#.KRB10134-5) and RANTES (cat#.KB1102) were purchased from Krishgen Biosystems, Mumbai, India and CCL1 (cat#.KTL11769) from (Kreative Technolabs, Los Angeles, CA, USA)

Techniques: Transfection, Migration, Activity Assay, Phagocytosis Assay, Mutagenesis

Journal: bioRxiv

Article Title: CD36: Hemin interaction axis to control immune responses and cytokine secretion from macrophages involving Lyn kinase

doi: 10.1101/2021.06.06.447270

Figure Lengend Snippet: (A) The validation of Lyn knockdown in macrophages. The macrophages either transfected with scrambled siRNA (sc siRNA) or Lyn siRNA and 48 h post transfection, lysates were probed with anti-Lyn antibody or anti-β actin antibody. The blot shows the 70-80% knockdown of Lyn protein. (B) Estimation of TNF-α levels from Lyn knockdown macrophage cell culture supernatants. The lyn knockdown macrophages treated with hemin showed reduced levels of TNF-α comparing to scrambled siRNA transfected macrophages indicates the hemin activates the cytokine signalling through CD36 and recruitment of lyn to the cytosolic domain of CD36. (C) Global cytokine profiling of Lyn knockdown macrophages. The macrophages were transfected with scrambled siRNA or Lyn target siRNA and either untreated or treated with hemin. The cytokine array was carried out on cell culture supernatants using mouse cytokine profiler array kit panel A. (D) The cytokines were normalized and plotted as fold change. Several cytokines such as CXCL-10, CXCL-1, MCP-1, CCL-3, CCL-4, CCL-2, and TNF-α levels were reduced (expressed in fold change) upon lyn knockdown. (E) The phagocytic activity of macrophages (with lyn knockdown) towards RBC. The macrophages either transfected with scrambled siRNA (sc siRNA) or or Lyn siRNA were either treated with hemin or remain untreated were incubated with normal RBC or oxidized RBC. Macrophages were stained with filipin to identify phagosomes containing engulfed material and observed under Nikon 80i fluorescence microscope in DAPI and TRITC filters to acquire filipin (Blue) and RBC (Red) fluorescence respectively. (F) The number of phagocytic macrophages were counted and plotted as number of phagocytic macrophages per 100 counted macrophages. The lyn knockdown in macrophages significantly reduced the non-opsonic phagocytosis of NRBC or oxiRBC in hemin treated condition. (G) Proposed hypothesis for hemin activated CD36 downstream signalling. The hemin interaction with CD36 induces the CD36 phosphorylation and sequestration of CD36 into intracellular vesicles. Further, the hemin binding to CD36 activates the intracellular signaling by recruitng the adaptor proteins from Src family to the cytoslic domain of CD36. The CD36-adaptor protein signalling complex intiates the cascade of events that trigger the transcription of pro-inflammatory cytokine genes.

Article Snippet: The cytokine ELISA kits for TNF-α (cat#. KRBA10602-5), MCP1 (cat#.KRB10134-5) and RANTES (cat#.KB1102) were purchased from Krishgen Biosystems, Mumbai, India and CCL1 (cat#.KTL11769) from (Kreative Technolabs, Los Angeles, CA, USA)

Techniques: Transfection, Cell Culture, Activity Assay, Incubation, Staining, Fluorescence, Microscopy, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Protective role of tenuigenin on sepsis-induced acute kidney injury in mice

doi: 10.3892/etm.2017.5164

Figure Lengend Snippet: Effect of TNG on inflammatory cytokine production. Levels of (A) TNF-α and (B) IL-6 in the kidneys were determined by ELISA. Values are expressed as the mean ± standard deviation (n=20). ##P<0.01 vs. the sham group; *P<0.05, **P<0.01 vs. the CLP group. TNF, tumor necrosis factor; IL, interleukin; CLP, cecal ligation and puncture; H, high-dose TNG (50 mg/kg); M, medium dose TNG (20 mg/kg); L, low-dose TNG (5 mg/kg); TNG, tenuigenin.

Article Snippet: Following centrifugation of the homogenate at 12,000 × g for 30 min at 4°C, the supernatant was collected and quantitatively assayed for TNF-α and IL-6 using tumor necrosis factor-α (cat. no. H052) and interleukin-6 (cat. no. H007) ELISA kits (Nanjing Jiancheng Biological Engineering Institute, Nanjing, China) according to the manufacturer's protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Ligation

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of imipenem combined with low-dose cyclophosphamide on the intestinal barrier in septic rats

doi: 10.3892/etm.2018.6373

Figure Lengend Snippet: TNF-α expression in the serum of each group. *P<0.05 vs. sham group; **P<0.05 vs. CLP group. CLP, cecal ligation and puncture; TNF, tumor necrosis factor; TX, cyclophosphamide.

Article Snippet: Interleukin (IL)-6 (cat. no. F15870), IL-10 (cat. no. F15900) and tumor necrosis factor (TNF)-α (cat. no. F3768; all Shanghai Westang Bio-tech Co., Ltd.) serum levels were detected using an ELISA according to the manufacturer's instructions.

Techniques: Expressing, Ligation

Journal: International Journal of Molecular Medicine

Article Title: Rutaecarpine ameliorates osteoarthritis by inhibiting PI3K/AKT/NF-κB and MAPK signalling transduction through integrin αVβ3

doi: 10.3892/ijmm.2023.5300

Figure Lengend Snippet: Primer sequences used in RT-qPCR.

Article Snippet: A primary antibody against p21 (cat. no. ab188224, 1:1,000) was acquired from Abcam. p16 (INK4A) antibody (cat. no. A0262, 1:750) was purchased from ABclonal Biotech Co., Ltd. Rabbit antibodies against TNF-α (cat. no. 48136) and IL-6 (cat. no. 32064) were acquired from Signalway Antibody and used at a 1:1,000 dilution.

Techniques: Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Rutaecarpine ameliorates osteoarthritis by inhibiting PI3K/AKT/NF-κB and MAPK signalling transduction through integrin αVβ3

doi: 10.3892/ijmm.2023.5300

Figure Lengend Snippet: Protective effects of RUT against IL-1β-induced inflammation and extracellular matrix degradation in mouse chondrocytes. The cells were exposed to IL-1β (5 ng/ml) with/without RUT (1, 2.5, 5 and 10 μ M) for 24 h. (A and B) The protein expression levels of MMP3, MMP13, IL-1β, COX2, IL-6, TNF-α, SOX9, COL II and aggrecan were determined using western blot analysis. (C-G) Quantification analysis of the results of western blotting. (D and E) Relative mRNA expression levels of IL-6 and TNF-α in chondrocytes stimulated with IL-1β (5 ng/ml) and treated with or without RUT (1, 2.5, 5 and 10 μ M) for 24 h. The values are presented as the mean ± SD of three independent experiments. # P<0.05 vs. control group; ** P<0.01, *** P<0.001 and **** P<0.0001 vs. IL-1β group. (H) The protein expression of COL II was detected using immunofluorescence following treatment of the cells with IL-1β (5 ng/ml) with/without RUT (10 μ M) for 24 h. IL-1β, interleukin 1 β; RUT, rutaecarpine; MMP, matrix metalloproteinase; COX2, cyclooxygenase 2; SOX9, SRY-box transcription factor 9; COL II, collagen type II.

Article Snippet: A primary antibody against p21 (cat. no. ab188224, 1:1,000) was acquired from Abcam. p16 (INK4A) antibody (cat. no. A0262, 1:750) was purchased from ABclonal Biotech Co., Ltd. Rabbit antibodies against TNF-α (cat. no. 48136) and IL-6 (cat. no. 32064) were acquired from Signalway Antibody and used at a 1:1,000 dilution.

Techniques: Expressing, Western Blot, Immunofluorescence