Extended Data Fig. 5: SDH inhibition causes ROS accumulation in IECs.
From: Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology
(a-d) PCECs were treated with DMSO, malonate or itaconate for 6 hours (n = 5). Cytoplasmic ROS measured by CellROX staining. (a) Representative images and (b) CellROX positive cells are shown. Mitochondria ROS measured by MitoSOX staining. (c) Representative images and (d) MitoSOX positive cells are shown. (e) PCECs were treated with DMSO or atpeninA5 for 4 hours. Cell death (left), CellROX (middle) and MitoSOX (right) levels were determined (n = 5). (f, g) Colon and ileum IECs were isolated from HCT recipients (BALB/c → C57BL/6). CellROX, MitoSOX (f) and mitochondrial membrane potential (ψm) (g) levels in (n = 5). (h, i) HCT settings are described in (f). Recipients orally received succinate every other day from day0 post HCT. Survival (h) and GVHD severity (i) are shown (n = 5). (j) Immunoblot of SUCNR1 in macrophage (MF, IC-21 cells) and IECs from BMT recipients 7days post HCT. Three independent experiments were performed. (k) Transcriptome analysis of IECs from recipients 7days post HCT. The mRNA expression of Hif1a and Cybb (CytochromeB). (n = 3). (l) Representative images of Hypoxyprobe in colon and ileum from recipients 7days after HCT (scale bar= 50 µm). Four independent experiments were performed. (m) Immunoblot image and protein density of ClpP in IECs from recipients 21days post HCT (Syn: n = 4, Allo n = 5). (n) Transcriptome analysis of IECs from recipients 7days post HCT. The mRNA expression of Clpp (n = 3). Data (f, g, k, l, m, n) are from biologically independent animals. Three independent experiments (a-e) were performed. All statistical analysis by one-way ANOVA analysis with Tukey post hoc test (b, d, e), two-tailed unpaired t-test (f, g), log-rank test (h) or two-tailed Mann-Whitney test (m) (mean ± s.e.m.). The p-values of the transcriptome analysis (k, n) obtained from DESeq-analysis.
