Fig 1: In vitro validation of the in silico–predicted docking site of TIMP-1 with CD74.A, representative confocal image of CD74-overexpressing LX-2 cells stimulated with fluorescently labeled recombinant human N-TIMP-1 (Alexa555N-TIMP-1). The nucleus is shown in blue, Alexa555N-TIMP-1 is shown in magenta, CD74 is shown in green. Regions of N-TIMP-1–CD74 colocalization are shown in gray. The scale bars represent 4 μm. B, representative dot blot assay showing the binding of endogenous CD74 derived from nondenatured B lymphoma cell lysates (shNT or shCD74) to immobilized TIMP-1 or MIF, respectively. Binding of cell lysate–derived CD74 to uncoated (shNT: n = 10, shCD74: n = 5), TIMP-1-coated (shNT: n = 10, shCD74: n = 5), and MIF-coated (shNT: n = 9, shCD74: n = 5) dots was determined by immunoblotting against CD74 (left) and quantified using densitometric analysis of dots (right). C, representative confocal image of a B lymphoma cell stimulated with fluorescently labeled recombinant human TIMP-1 (Alexa555TIMP-1). Distribution of TIMP-1 (magenta), CD74 (green), as well as TIMP-1–CD74 colocalization (white) is shown. Surfaces of TIMP-1–CD74 colocalization are displayed in gray, and the nucleus is shown in blue. The scale bars represent 2 μm. D, representative dot blot assay investigating N-TIMP-1 binding to CD74. Immobilized N-TIMP-1 was incubated with nondenatured cell lysate from B lymphoma cells. Binding of cell lysate-derived CD74 to uncoated (n = 5) and N-TIMP-1-coated dots (n = 5) was determined by immunoblotting against CD74 (left panel) and quantified using densitometric analysis of dots (right panel). B and D, results are represented as the mean ± SD. For statistical analyses, a one-sample t test was used in case of normal distribution or a one-sample Wilcoxon test in absence of normal distribution, ∗p ≤ 0.05. MIF, macrophage migration inhibitory factor; N-TIMP-1, N-terminal domain of TIMP-1; TIMP-1, tissue inhibitor of metalloproteinases-1.
Fig 2: TIMP-1–CD74 interaction induces ZAP-70 signaling.A, C, D, and E, representative Western blots of cell lysates from B lymphoma cells treated as indicated. Levels of phosphorylated ZAP-70 and GAPDH were analyzed. A, cells were treated with 500 ng/ml human recombinant WT TIMP-1 (n = 3), 100 ng/ml human recombinant MIF (n = 6) or were left untreated (n = 4) (left). Fold change of ZAP-70 activation was determined by densitometric analysis of band intensities normalized to levels of GAPDH (right). B, representative flow cytometric analysis of CD74 expression by three different CD74 knockdown B lymphoma cells (shCD74 #1, #2, #3) or control cells (shNT). Contour plots show the frequency of CD74-positive cells. C, representative Western blots of cell lysates from three different CD74 knockdown B lymphoma cells (shCD74 #1, #2, #3) or control cells (shNT) treated as indicated. D, B lymphoma cells were stimulated with 500 ng/ml human recombinant TIMP-1 (n = 5), equimolar human recombinant N-TIMP-1 (n = 4), or left untreated (n = 5) (left). Fold change of ZAP-70 activation was determined by densitometric analysis of band intensities normalized to levels of GAPDH (right). E, B lymphoma cells were stimulated with 500 ng/ml TIMP-1 after preincubation with or without an anti-CD74 blocking peptide. A, C, and D, results are represented as the mean ± SD. For statistical analyses, a one-sample t test was used in case of normal distribution or a one-sample Wilcoxon test in the absence of normal distribution, ∗p ≤ 0.05; n.s., not significant. MIF, macrophage migration inhibitory factor; N-TIMP-1, N-terminal domainof TIMP-1; TIMP-1, tissue inhibitor of metalloproteinases-1; ZAP-70, zeta chain–associated protein kinase-70.
Fig 3: ChitoAntibac PDMS enabled fast release of MIF and induced distant macrophage recruitment. (A) Load efficiency of MIF for ChitoAntibac PDMS implants with various crosslinking conditions was assessed after 2 h of loading. ChitoAntibac PDMS #4 provided the highest MIF loading and therefore was chosen for subsequent experiments. (B) MIF loading efficiency using ChitoAntibac PDMS as a function of time. Maximal loading rate was indicated in the plot. (C) Time-dependent MIF release profile using ChitoAntibac PDMS#4 at physiological temperature (37 °C). (D) Schematic illustrating the experimental setup to investigate the effect of MIF released from the ChitoAntibac PDMS implants on macrophage recruitment. (E) Representative images showed macrophage across different groups migrated towards the other side of the inserts or the cell culture well plates after 24 h incubation. Scale bar = 100 μm. (F) Cell counts of migrated macrophages across different groups. Data are presented as mean ± SD. Triplicate experiments were performed. Statistical significance is indicated in the plots where necessary, with * denotes p < 0.05 and ** denotes p < 0.01.
Fig 4: ChitoAntibac PDMS loaded with MIF activated M1 polarization to enhance bacterial phagocytosis. (A) Percent of M1 macrophages after treatment with MIF-loaded ChitoAntibac PDMS for various durations. Quantification of M1 macrophage population was performed by counting cells that had CD 80 expression level more than 2-fold of that of the cells in the control group. (B) Representative fluorescent images showed macrophages stained with CD 80 antibody (green) after treatment with MIF-loaded ChitoAntibac PDMS for various durations. Scale bar = 50 μm. (C) Representative fluorescent images showed phagocytosis of S. aureus in macrophages with various pretreatments post 3 h of bacterial inoculation. Macrophages were counterstained with Hoechst 33342 (blue) and Phalloidin (red) with the endocytosed bacteria displayed in green. Scale bar = 20 μm. (D) Number of S. aureus internalized by macrophages with different pretreatments. n = 40. Triplicate experiments were performed. Statistical significance is indicated in the plots where necessary, with * denotes p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 5: ChitoAntibac PDMS loaded with phage K exhibited rapid and efficient removal of S. aureus in vitro. (A) Optical density measurement showed dose- and time-dependent bacteria-eliminating property of phage K in vitro. (B) Quantification of live bacteria after treatment with different concentrations of phage K. MIC99 was indicated in the plot and used as a baseline to determine the effective antibacterial dose range (highlighted in light brown). (C) Quantification of phage K loaded into ChitoAntibac coated PDMS sheets and Ti alloy. (D) Optical density measurement shows the antibacterial efficacy of phage-loaded ChitoAntibac PDMS and Ti alloy as a function of time. (E) Log reduction of live bacteria (S. aureus) after treatment with ChitoAntibac coated PDMS sheets and Ti alloy. Insets showed optical images of bacterial colonies formed on agar plates that corresponded to respective treatment. (F) Cell viability of mouse neurons after 1-, 3-, 5-, 7-day treatment with ChitoAntibac PDMS loaded with MIF or Phage K. (G) Cell viability of MCC3T3-E1 bone cells after 1-, 3-, 5-, 7-day treatment with ChitoAntibac Ti loaded with MIF or Phage K. Triplicate experiments were performed. Statistical significance is indicated in the plots where necessary, with ** denotes p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Supplier Page from Abcam for Recombinant Human MIF protein