Fig. 7. Pyridoxal and supersulfides reverse enhanced inflammatory response of macrophages in prolonged hypoxia.
a, Lysosomal acidification in BMDMs. Representative AcidiFluor ORANGE staining (left) and its quantification (right) from three independent experiments. CHyp + Pyridoxal, BMDMs differentiated under 1% oxygen in the presence of 50 μg ml−1 pyridoxal. b. Immunoblot analysis detecting Lamp1 in BMDMs. Tubulin was detected as a loading control. A representative result from three independent experiments is shown. c, Expression of representative lysosome-related genes in BMDMs (n = 3 biologically independent samples). d, Immunoblot analysis detecting 2OG-dependent dioxygenases in BMDMs. Tubulin was detected as a loading control. A representative result from three independent experiments is shown. e, Relative Il6 expression in BMDMs (n = 3 biologically independent samples). f, ELISA of IL-6 in the culture supernatant of BMDMs (n = 4 biologically independent samples). g, Il6 expression in BMDM with or without PNPO overexpression (n = 4 biologically independent samples). h, SSP4 staining to detect supersulfides in BMDM. Representative SSP4 staining (left) and quantification (right) from three independent experiments. i, Effects of GSSSG, a supersulfide donor, and GSSG on lysosomal acidification. Representative AcidiFluor ORANGE staining (left) and its quantification (right) from three independent experiments. Oxidized glutathione, GSSG, was added as a negative control. j, Il6 expression in BMDM treated with GSSSG or GSSG (n = 3 biologically independent samples). Scale bars, 50 μm (a,h,i). Error bars represent s.e.m. (c,e–g,j). One-way ANOVA (a,h) and two-way ANOVA (c,e–g,i,j) were conducted to evaluate statistical significance.