Figure 3. Role of Csf3 in macrophage activation after treatment with PA. To validate the results of RNA sequencing, the Csf3 mRNA level in macrophages was determined after PA treatment. J774 cells were incubated with PA and then treated with or without LPS. The cells were harvested and the relative expression of Csf3 was measured using qPCR and normalized to Gapdh expression (A). Silencing of Csf3r attenuated the PA-induced increased secretion of chemokines in LPS-stimulated macrophages. J774 cells were transfected with siRNA against Csf3r prior to stimulation of the cells with PA with or without LPS. In the culture media, the levels of chemokines were determined using ELISA. Experiments were conducted with technical duplicates, and data presented are based on 3 independent replicates.

ELISA = enzyme-linked immunosorbent assay; IL = interleukin; LPS = lipopolysaccharide; mmLDL = minimally modified low-density lipoprotein; mRNA = messenger RNA; PA = palmitate; qPCR = quantitative polymerase chain reaction; siRNA = small interfering RNA.

^*p<0.05, ^†p<0.01.