Fig. 2: Immunophenotyping of Tgm2 CRISPR-Silenced BMMs.

Bone marrow was isolated from C57BL/6 J mice and cultured in the presence of M-CSF for BMMs differentiation for 4 days. BMMs were then transfected with CRISPR targeting Tgm2 and evaluated 72 h post-transfection. A Western blot analysis confirming Tgm2 silencing in sgControl (n = 9) or sgTGM2 (n = 9) transfected BMMs. B Evaluation of TGM2 secretion in CRISPR silenced BMMs (n = 8/group), C Transglutaminase enzymatic activity in Tgm2 CRISPR silenced BMMs (n = 7/group) and D Cytokine profile in harvested supernatant from CRISPR targeted BMMs treated with LPS for 24 h (n = 9–10/group). E Gating strategy for flow cytometric analysis of CRISPR silenced BMMs (n = 10/group). Flow cytometric data analysis from CRISPR silenced BMMs polarized into E M0, F M1 (50 ng/mL IFNy and 10 ng/mL LPS in DMEM) or G M2 (M2 = 20 ng/mL IL-4 in DMEM), polarizing stimuli, on day 7 of differentiation. Cells were assessed 24 h post-polarization. Data are shown as bar graphs with SEM of seven or ten mice per control or treated group and are representative of three independent experiments. For all graphs, data normality status was used to determine statistical analysis. Normal data statistical significance was determined by parametric student’s unpaired t-test, while not normally distributed data was analyzed using non-parametric Mann-Whitney test. ns = P > 0.05, **=P < 0.05, ** =P < 0.01, ***P < 0.001.