Fig 1: Alterations in the gene expression of H2S-modulating enzymes in the course of spontaneous (contact inhibition-induced) differentiation of Caco-2 cells to a colonocyte-like phenotype. Caco-2 cells were cultured for the indicated periods of time with respect to day 0, i.e., the day they reached confluence (PC, days post-confluence). Relative mRNA levels of SELENBP1, MPST, CBS, CTH and SQOR at different time points during spontaneous differentiation (as compared to the reference point PC 0) were determined by qRT-PCR, with normalization against the housekeeping gene RPLP0. Differentiation of the Caco-2 cells was substantiated by an increase in mRNA levels of ALPI. Three independent experiments were performed; the data represent means ± SD. Statistical analysis was done using the Friedman test and Dunn’s post-hoc test, with statistical significance at p < 0.05 (*).
Fig 2: Spontaneous and butyrate-induced differentiation affect the protein levels of H2S-modulating enzymes in Caco-2 cells in a partially divergent manner. Proliferating and spontaneously differentiated cells were treated with sodium butyrate for 72 h until either day 1 (PC 1) or day 14 (PC 14) of differentiation. (A–E) Relative protein levels of SELENBP1, CBS, CTH, MPST and SQOR, respectively, in cell lysates were determined by immunoblotting and densitometric analysis of the blots, with normalization against Ponceau S-stained protein bands (the untreated control at PC 1 was set as reference). Three independent experiments were performed; the data represent means ± SD (upper panels). Representative immunoblots, with corresponding detection of the housekeeping protein GAPDH after membrane stripping, are shown (lower panels). (F) The relative enzymatic activity of ALPI in the lysates (the untreated control at PC 1 was set as reference) was measured photometrically to monitor differentiation. Statistical analysis was done using the Friedman test and Dunn’s post-hoc test, with statistical significance at p < 0.05 (*) and p < 0.01 (**).
Fig 3: Overview of H2S biogenesis in the mammalian colon. Gut microbiota generate H2S by fermentation of dietary sulfur-organic compounds and by sulfate reduction (as well as butyrate by fermentation of dietary fiber). H2S production in colonic epithelial cells occurs through the four enzymes CBS, CTH, MPST and SELENBP1. Sulfur-containing amino acids serve as precursors for H2S production by CBS, CTH and MPST, while the SELENBP1 substrate methanethiol is produced from methionine by colonic microbiota. SQOR is a key enzyme in H2S catabolism. Scheme created with Biorender.com.
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