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Figure 7 | Veterinary Research

Figure 7

From: FABP4-mediated lipid droplet formation in Streptococcus uberis-infected macrophages supports host defence

Figure 7

Inhibiting FABP4 expression leads to reduced LDs and increased bacterial burden. RAW264.7 cells were treated with 40 µM BMS309403 for 24 h or transfected with 50 nM siFabp4 for 24 h at 37 ℃ using Lipofectamine 3000 reagent (Invitrogen) to inhibit FABP4 expression prior to infection with S. uberis in mid-exponential phase (MOI = 10) for 3 h at 37 ℃. A Fabp4 mRNA expression in the control and interference groups during S. uberis infection. B Protein expression levels of FABP4 with or without BMS309403 or siFabp4 during S. uberis infection were determined by Western blots of RAW264.7 cells. For quantitative analysis, bands were evaluated densitometrically with ImageJ analyser software normalized for β-actin density. C Relative mRNA expression of genes related to the synthesis of fatty acids. D Flow cytometry of BODIPY-stained RAW264.7 cells. Left: Representative histograms. Right: Mean fluorescence intensities from 3 independent experiments. The dotted line indicates background fluorescence in uninfected cells. Data are presented as the means ± SEMs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA. E Immunofluorescence and LD staining was performed by FABP4 (red) and BODIPY (green) in the control group and stimulated S. uberis groups as well as the chemical pretreatment groups. Images are representative of n = 10 samples/group. Scale bar, 20 µm. F Relative Nos2 and Cxcl9 mRNA expression. G Tnf, Il1b, and Il6 mRNA expression. H The number of S. uberis colonies in the RAW264.7 cells after FABP4 or Fabp4 treatment on THB agar plates. Data are presented as the means ± SDs (n = 6). *(P < 0.05) = significantly different compared with the S. uberis groups by one-way ANOVA. A–D and F–H experiments were repeated 3 times. Data are presented as the means ± SEMs (n = 6). *(P < 0.05) = significantly different between the indicated groups by one-way ANOVA.

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