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. 2019 Mar 12;9:4246. doi: 10.1038/s41598-019-38982-0

Figure 3.

Figure 3

Rv3463 induces macrophage activation via TLR2 and TLR4. Bone marrow-derived macrophages (BMDMs) derived from wild-type (WT), TLR2−/−, TLR4−/−, and TLR2/4−/− mice were treated with Rv3463 (5 μg/ml), lipopolysaccharide (LPS, 100 ng/ml), or Pam3CSK4 (100 ng/ml) for 24 h. (A) The production of TNF-α, IL-6, and IL-12p70 in the culture supernatants was determined by ELISA. All data are expressed as mean ± SD (n = 3). (B) Expression of CD80 and MHC class II molecules on BMDMs stimulated with each antigen was determined by staining and flow cytometry. The bar graphs show the mean percentage ± SEM of each surface molecule on F4/80+ cells across three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 for treatment values in BMDMs from TLR2−/−, TLR4−/− or TLR2/4−/− mice compared to those of Rv3463-, LPS- or Pam3CSK4-treated BMDMs from WT mice. (C) Fluorescence intensities of anti-Rv3463 bound to the surface of BMDMs. BMDMs derived from WT, TLR2−/−, TLR4−/−, and TLR2−/− mice were treated with Rv3463 (5 μg/ml) for 1 h, fixed, and then stained with DAPI (blue) and Alexa488-conjugated anti-Rv3463 antibody. Representative images out of three independent experiments are shown. Scale bar, 10 μm. ***p < 0.001 for difference between BMDMs from each mice. (D) The cell lysates from BMDMs treated with Rv3463 for 6 h were used for immunoprecipitation with anti-mouse IgG and anti-His (upper), or anti-TRL2 and TLR4 antibodies (lower). Thereafter, proteins were detected using immunoblotting with anti-His, anti-TLR2 or anti-TLR4 antibodies. The total is shown as the mean total cell lysates (input).