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. 2020 Oct 8;10:16802. doi: 10.1038/s41598-020-73912-5

Figure 3.

Figure 3

TNF-α mediated pro-inflammatory monocytic responses requires nSMase2. Primary monocytes and THP-1 monocytes were transfected with scramble-siRNA (negative control; Sc) or SMPD3 siRNA and incubated for 36 h. Real-time PCR was performed to measure (A) SMPD3 expression in primary monocytes and (B) CD11c was determined by real time RT-PCR. Cells were stained with antibodies against CD14 and CD11c along with matched isotypes and were subjected to flow cytometry analysis. (C) Flow cytometry data are presented as a bar graph of mean staining index of CD14+CD11c+ cells as well as (D) representative histogram. nSMase deficient THP-1 cells were treated with TNF-α and vehicle. (E) SMPD3 expression in transfected THP-1 monocytic cells. (F) CD11c mRNA was determined by real time RT-PCR. Cells were stained with antibody against CD11c along with matched isotype controls and assessed by flow cytometry. (G) Flow cytometry data are presented as a bar graph of mean staining index as well as (H) representative histogram. (I–J) Secreted IL-1β and MCP-1 by nSMase2 deficient cells. The results obtained from minimum three independent experiments with three replicates of each experiment are shown. All data are expressed as mean ± SEM (n ≥ 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 versus vehicle.