Fig 1: NPWT materials do not require direct contact with cells to suppress inflammatory activation. (a) Schematic of the transwell experiments, explaining the indirect culture setup and the workflow. (b) ELISA results of the transwell culture experiments for TNF-a and MCP-1 production by the macrophages. (c) qRT-PCR results of the transwell culture experiments for TNF, CCL2, and IL1B by the macrophages. RPL37A was used a housekeeping gene for normalization. Both experiments used empty transwell (no material) controls. All bar plots are represented as the mean of 3 independent experiments, with error indicating the S.E.M. Abbreviations are as follows: VC—Veraflo Cleanse, GF—Granufoam, CT—Cotton gauze, Ctrl—No material control. * indicates p < 0.05 as tested using one-way ANOVA with multiple comparisons.
Fig 2: Effects of C29 and TAK-242 on Pam- or LPS-induced pro-inflammatory reactions in RAW264.7 macrophages. (A) Effects of C29 and TAK-242 on Pam- or LPS-induced NF-?B activation in RAW264.7 macrophages. (B) Effects of C29 and TAK-242 on Pam-elevated supernatant IL-6, MCP-1 and TNF-a in RAW264.7 macrophages. (C) Effects of C29 and TAK-242 on LPS-elevated supernatant IL-6, MCP-1 and TNF-a in RAW264.7 macrophages. Mean ± SD (n = 3). ##P < 0.01 vs. negative control; *P < 0.05 and **P < 0.01 vs. Pam or LPS alone. ND, not detected.
Fig 3: Comparison of the effects of trimer WT-S and Omicron-S on inflammatory responses in RAW264.7 macrophages. (A) Effects of trimer WT-S and Omicron-S on NF-?B activity. The cells stably transfected with NF-?B-TA-luc plasmid were stimulated with trimer WT-S or Omicron-S at the indicated concentrations. Four hours later, the luciferase activities were detected. (B–E) Effects of trimer WT-S and Omicron-S on supernatant TNF-a, MCP-1, IL-6 and IFN-ß in RAW264.7 macrophages. Cells were treated with trimer WT-S or Omicron-S at the indicated concentrations. Twenty-four hours later, supernatant TNF-a, MCP-1, IL-6 and IFN-ß were determined. LPS was used as a positive control for INF-ß. Mean ± SD (n = 3). **P < 0.01 vs. 0 nM of WT-S (negative control); #P < 0.05 and ##P < 0.01 vs. 0 nM of Omicron-S (negative control); ??P < 0.01. ND, not detected.
Fig 4: Macrophages display lower inflammatory polarization on the NPWT materials during direct culture. (a) Schematic of the direct culture experiments, explaining the direct culture setup and the workflow. (b) ELISA results of the direct culture experiments for TNF-a, and MCP-1 production by the macrophages. (c) qRT-PCR results of the direct culture experiments for TNF, CCL2, and IL1B by the macrophages. RPL37A was used a housekeeping gene for normalization. (d) Schematic depicting the various possible biochemical and biomechanical interactions between the material surfaces and the cells. All bar plots are represented as the mean of 3 or 6 independent experiments, with error indicating the S.E.M. Abbreviations are as follows: UL—Ultralow attachment plate surface, VC—Veraflo Cleanse, GF—Granufoam, CT—Cotton gauze, GL—Glass coverslip. * indicates p < 0.05 as tested using one-way ANOVA with multiple comparisons.
Fig 5: Effects of TAK-242 and C29 on the pro-inflammatory reactions induced by two trimer S-proteins. (A–B) Effects of TAK-242 and C29 on supernatant TNF-a, MCP-1 and IL-6 induced by WT-S (A) or Omicron-S (B) in RAW264.7 macrophages. The cells were treated with trimer WT-S or Omicron-S (2.4 nM) in the presence of TAK-242 (1 µM) or C29 (40 µM). Twenty-four hours later, supernatant TNF-a, MCP-1 and IL-6 were determined. ND, not detected. (C) Effects of TAK-242 and C29 on trimer WT-S- or Omicron-S-induced NF-?B activation. RAW264.7 cells stably transfected with NF-?B-TA-luc plasmid were treated with C29 (40 µM) or TAK-242 (1 µM) and then stimulated by trimer WT-S or Omicron-S (2.4 nM). Four hours later, the luciferase activities were detected. Mean ± SD (n = 3). ##P < 0.01 vs. negative control; **P < 0.01 vs. WT-S or Omicron-S alone.
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