Multi-Walled Carbon Nanotubes Induce Arachidonate 5-Lipoxygenase Expression and Enhance the Polarization and Function of M1 Macrophages in vitro

Data Collection Methods

1. Characterization of nanoparticles

• • MWCNTs (Mitsui-7) were prepared in a control medium (CM) [Dulbecco’s modified eagle’s medium (DMEM) with 1% fetal bovine serum (FBS)] at a concentration of 2 mg/ml through vortex and by sonication.
  • Carbon black (CB, Printex 90) was prepared in a control medium (CM) like MWCNTs and used as carbon-based particle control.
  • Stock solutions of MWCNTs or CB were further diluted with the culture media for MWCNTs at 2.5 or 10 µg/ml or for CB at 2.5, 10, or 30 µg/ml, sonicated immediately before use, and were treated for indicated time.

2. Cell culture, polarization, and treatment

• • J774A.1 murine monocyte/macrophage cells were grown in DMEM with 10% fetal bovine serum.
  • J774A.1 cells (5 x 10⁵ cells/ml) were seeded in DMEM with 3% FBS for 1 day and then treated with other reagents for 1 day or 3 days.
  • M1 polarization was induced with interferon γ (20 ng/ml) plus lipopolysaccharides (100 ng/ml) and M2 polarization was induced with interleukin 4 (20 ng/ml) for indicated time (typically three days).
  • Control media (media with 1% FBS) were prepared and treated to establish a negative control response.
  • HL-60, a promyelocytic cell line, was grown in the RPMI1640 medium with 10% FBS.
  • Differentiated HL-60 cells (dHL-60) were prepared with use of 2 µM all-trans retinoic acid (ATRA) treatment for 3 days.

3. Quantitative real-time PCR (RT-qPCR)

• • Detection and quantification of Alox5 or β-actin at mRNA level.
  • Real-time qPCR was performed, and the relative expression change values were calculated as 2^−ΔΔCt and expressed as fold change in comparison with untreated control.

4. Immunoblotting

• • Detection and quantification of Alox5, CD68, Alox5ap, or β-actin.
  • Images were scanned using HP scanjet and were used to quantify each band with ImageJ software.

5. Detection of nitric oxide synthase 2 (Nos2, mouse iNOS) expression

• • Detection and quantification of Nos2 expression using an intracellular Nos2 detection assay kit.
  • The fluorescence signal was measured using a fluorescence plate reader and the relative fluorescence unit (RFU) was expressed.

6. Enzyme-linked immunosorbent assay (ELISA)

• • Production of proinflammatory cytokines (TNF-α, IL-1β) and lipid mediators (LTB4, PGE2) in cell-free culture supernatant were measured using ELISA kits with a microplate reader equipped with SOFTmax PRO 4.0.

7. Chemotaxis assay

• • Transwell cell migration assay was performed using a cell migration assay kit to determine the effect of MWCNTs on neutrophilic cell migration in vitro.
  • ATRA-induced differentiated HL-60 (dHL-60) cells were collected and suspended in RPMI 1640 medium without FBS (2.5×10⁵ cell/well/100 µl) and plated on each upper chamber.
  • The lower chambers were filled with cell-free culture supernatants, indicated amount of MWCNTs in the basal RPMI 1640 medium containing 1% FBS, or RPMI 1640 medium containing 10% FBS.
  • For inhibition assay, a cell-free culture media from cells treated with a specific cyclooxygenase 2 inhibitor, NS-398 (at 2 or 10 µM), a leukotriene A4 hydrolase inhibitor, Acebilustat (at 1 or 5 µM), or dimethyl sulfoxide (DMSO) as a vehicle 6 hr prior to MWCNTs or IFN-γ+LPS exposure added at each lower chamber. A specific LTB4 receptor inhibitor LY293111 (5 or 25 nM) or DMSO as a vehicle was added directly into the dHL-60 cell suspension.
  • The fluorescence signal was measured using a fluorescence plate reader and the relative fluorescence unit was expressed to show the migrated cells.

8. Alox5 gene silencing

• • To knockdown Alox5 gene expression in macrophages, mouse Alox5-specific short hairpin (shRNA) as RNA interference was introduced into macrophages and incubated for 2 days and then cells were used for RT-qPCR and immunoblotting analysis.
  • Cells were treated with MWCNTs or M1 inducer and subsequently used for RT-qPCR and immunoblotting analysis, and the cell-free culture media was collected and used for ELISA assay.