Skip to main content
. 2021 Dec 2;12:773013. doi: 10.3389/fimmu.2021.773013

Figure 7.

Figure 7

TP4-induced TNF-α-stimulated gene 6 (TSG-6) secretion from VK2 cells is required for STAT6 activation in THP-1/GV cells. (A) Detection of phosphorylated NFκB p65 subunit (p-p65), STAT3 (p-STAT3), and STAT6 (p-STAT6) after 1 h treatment with conditioned medium (CM) from TP4 (3.91 or 7.82 μg/ml)-treated VK2 cells to THP-1/GV cells by immunoblotting. GAPDH was used as an internal control to show equal protein loading. (B) TSG-6 expression in VK2 cells after TP4 (3.91 or 7.82 μg/ml) treatment for 6 h (left) or 7.82 μg/ml TP4 treatments for 0, 4 or 6 h (right), measured by qRT-PCR. Data were represented the normalized target gene amount relative to the group of 0 μg/ml. (C) IL-4 expression in VK2 cells after TP4 (3.91 or 7.82 μg/ml) treatment for 6 h (left) or 7.82 μg/ml TP4 treatments for 0, 4 or 6 h (right), measured by qRT-PCR. Data were represented the normalized target gene amount relative to the group of 0 μg/ml. (D) Detection of TSG-6 expression in VK2 cells after 6 h treatment of TP4 (1.95, 3.91, 7.82 or 15.63 μg/ml) by immunoblotting. α-tubulin was used as an internal control to show equal protein loading. (E) Detection of TSG-6 expression in VK2 cells after 7.82 μg/ml TP4 treatment for 0, 2, 4, 6, or 8 h by immunoblotting. α-tubulin was used as an internal control to show equal protein loading. (F) The CM from vehicle (Veh; PBS)- or TP4-treated VK2 cells were incubated with TSG-6-neutralizing antibody (anti-TSG6) or IgG control for 1 h and then used to treat THP-1/GV cells. After incubation, p-STAT6 in THP-1/GV cells was detected. GAPDH was used as an internal control to show equal protein loading. (G) Macrophage surface marker (CD206) was detected by flow cytometry after CM incubation. The mean fluorescence intensity (MFI) is shown. The black bar graph indicates isotype controls. (H) Gene expression levels of ARG1, YM-1, and RELMα were measured after treatments by qRT-PCR. Data were represented the normalized target gene amount relative to the vehicle-treated group (lane 1). Data are presented as mean ± SD of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not statistically significant).