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. 2021 Jan;64(1):115–125. doi: 10.1165/rcmb.2020-0360OC

Figure 4.

Figure 4.

Lactate induces histone lactylation in macrophages. (A) BMDMs were treated with or without 20 mM lactate for 24 hours. The cells were lysed, and lactate contents in the cellular lysates were determined (n = 3; mean ± SD). ***P < 0.001 by two-tailed Student’s t test. (B) BMDMs were treated with or without 20 mM lactate as in A. Concentrations of the indicated proteins in whole cell lysates or HCl-extracted histones were determined by Western blotting. (C) BMDMs were treated with or without lactate. The entire blot was developed for lactyl-lysine. (D) BMDMs were treated with conditioned media from normal fibroblasts or TGF-β1–induced myofibroblasts. Twenty-four hours after treatment, cells were lysed, and lactate contents in the cellular lysates were determined (n = 3; mean ± SD). *P < 0.05 by two-tailed Student’s t test. (E) BMDMs were treated with conditioned media from normal fibroblasts or TGF-β1–induced myofibroblasts as in (D). Concentrations of the indicated proteins were determined by Western blotting. (F) C57BL/6 mice were i.t. instilled with saline or BLM. Three weeks after treatment, mice were killed, and lung slices were prepared. The expression of F4/80 and lactyl-histone were evaluated by immunofluorescence staining and fluorescence microscopy. (G) Slices of normal con and idiopathic pulmonary fibrosis lungs were prepared. The expression of CD68 and lactyl-histone were evaluated by immunofluorescence staining and fluorescence microscopy. (H and I) C57BL/6 mice were i.t. instilled with saline or BLM. Three weeks after treatment, BALFs were harvested, and alveolar macrophages were isolated and cultured on coverslips. The expression of F4/80 and lactyl-histone were evaluated by immunofluorescence staining and fluorescence microscopy (H). Single-cell fluorescence intensity was measured by ImageJ for ∼100 randomly selected cells from each group, and the average was calculated. ***P < 0.001 by two-tailed Student’s t test. The box-and-whisker plots depict the 25th and 75th percentiles and the median, minimum, and maximum values (I). (F–H) Nuclei were counterstained with DAPI. Scale bars, 100 μm. AE = HCl-extracted; IPF = idiopathic pulmonary fibrosis; MFI = mean fluorescence intensity; WCE = whole cell lysates.