Fig 2: The CD3 expression on RAW cells is modulated by time and nutrient deficiency. Flow Cytometry evaluated RAW cells for up to 72 hours for CD3 (A). Relative expression of CD3 was assessed by qPCR for up to 72 hours (B), and the relative fold change across time is shown (C). Other cultures were maintained under FBS restriction or pyruvate deprivation for 24 hours to assess changes in CD3 expression at both the protein level by flow cytometry (D) and the transcriptional level by qPCR (E). The live cell percentage of these cultures was determined by flow cytometry (F). Additionally, TNF levels were quantified in the culture supernatant (G), and CCL5 and HIF1α transcriptional levels were evaluated by qPCR (H, I), respectively). Data shown were represented as mean ± SD (A-C, n=4; D-G, n=8); asterisks indicate the statistically significant difference between the compared conditions: ∗p < 0.05; **p < 0.01; ***p<0.001; ****p<0.0001.

Fig 3: Cytokine production of RAW cells after stimulation of the CD3 complex and Fc-γR. Cytokine levels were evaluated using a bead-based multiplex assay in the supernatants obtained from cell cultures with different experimental conditions: Unstimulated as control conditions (RPMI), stimulated with anti-CD3 + IgG2a (anti-CD3/IgG), isotype antibody controls (Isotype control), and a positive known stimulus such as Lipopolysaccharide (LPS) or PMA-Ionomycin (PMA/IO). Levels of the proinflammatory IFN-γ (A), TNF (B), and IL-6 (C); the antiinflammatory IL-4 (D), IL-5 (E), and IL-10 (F); and IL-2 (G). Data were shown as mean ± SD (n=8); asterisks indicate the statistically significant differences between the compared conditions: *p < 0.05; ** p < 0.01; *** p < 0.001.