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. 2021 Mar 31;12:640718. doi: 10.3389/fimmu.2021.640718

Figure 6.

Figure 6

PH-fibroblast conditioned media increased free NADH, as well as CtBP1 levels in BMDMs. MTOB, a small molecule inhibitor of CtBP1, inhibited gene activation in BMDMs in a fibroblast-macrophage co-culture system. (A) Fluorescence lifetime imaging (FLIM) of two-photon excited NADH was performed to directly visualize free-NADH (red: arbitrarily colored shorter excitation lifetime, free-NADH. Green: bound-NADH, longer excitation lifetime). A significantly increased detection of free-NADH was seen in PH-CM treated BMDM compared to untreated BMDMs. Data is represented as percentage of red signal and shown as mean ± sem. **p < 0.01 compared to untreated BMDMs. (B) NADH/NAD+ ratio was measured using Mass Spectrometry analysis and an enzymatic cycling reaction and represented by lactate/pyruvate ratio. *p < 0.05 compared to untreated BMDMs. (C) qPCR analysis showed significantly increased Ctbp1 levels in PH-CM treated compared to untreated BMDMs. Data is represented as fold change relative to untreated BMDMs and displayed as mean ± sem. ***p < 0.001. (D) Co-cultured PH-Fibs and BMDMs were treated with MTOB, a small molecule inhibitor of CtBP1. MTOB significantly decreased inflammatory genes Il1b, TLR2/4, IL6, metabolic genes Glut1, Ldha, Hk2, Arg1, vascular remodeling genes Tgm2, Thbs1, and transcriptional regulators and signaling genes mTOR, MyD88, HIF1a in co-cultured BMDMs. Data is represented as fold change relative to untreated BMDMs, and displayed as mean ± sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.