Fig. 3: ACOD1-deleted human iPSC-derived macrophages demonstrated enhanced pro-inflammatory activation.

a Comparison of the DNA sequence in the ACOD1 knockout iPSC clone (by Sanger sequencing) with the ACOD1 WT DNA sequence showed an 8 bp deletion in the sgRNA targeted region. b Western blotting for ACOD1 expression in WT and ACOD1^-/- iPSC-derived macrophages (iMAC) after LPS and IFN-γ stimulation for 24 h. This experiment has been repeated for three times with similar results. c Mass spectrometry quantification of the cellular itaconate (ITA) concentration in WT and ACOD1^-/- iMACs after LPS and IFN-γ stimulation for 24 h (WT, n = 6 biologically independent samples; ACOD1^-/-, n = 4 biologically independent samples). Statistics by unpaired t test. (P < 0.0001) d, e CD80 expression on WT and ACOD1^-/- iMACs and quantification was determined by flow cytometry under different treatments, including 100 ng/mL LPS or 50 ng/mL LPS plus 50 ng/mL IFN-γ stimulation for 24 h (e, n = 3 biologically independent samples). Statistics by two-way ANOVA test. (unstimulated, P = 0.8871; LPS, P < 0.0001; LPS + IFN-γ, P < 0.0001) f The levels of the indicated cytokines/chemokines in the medium of iMAC culturing were determined 24 h post IFN-γ and LPS challenge (n = 3 biologically independent samples). Statistics by unpaired t test. g Seahorse extracellular metabolic flux analysis of oxygen consumption rates (OCR). LPS and IFN-γ stimulated WT or ACOD1^-/- iMACs were sequentially treated with oligomycin (1.5 μM), fluorcarbonylcyanide phenylhydrazone (FCCP; 2 μM), and rotenone and antimycin A (0.5 μM each) (n = 3). h Basal OCR, maximal respiration capacity (MRC), and ATP production rate were calculated with Wave 2.4.0. (n = 3 biologically independent samples). Statistics by unpaired t test. c, e, f, g and h Data was shown as mean ± SD. Source data are provided as a Source Data file.