Fig. 4.

PPARβ/δ deficiency in fibroblasts lowers oxidative stress in colon cell lines. a, b FACS analysis of CD11b + cell numbers in colons from FSPCre-Pparb/d^−/− and Pparb/d^fl/fl mice treated with vehicle or DSS (a) and APC^min/+FSPCre-Pparb/d^−/− and APC^min/+Pparb/d^fl/fl colons (b). Fresh tissue biopsies were dissociated using the gentleMACS Dissociator according to manufacturer’s instruction. Homogenates were strained, washed, and processed for staining with fluorophore-conjugated antibodies on ice. Flow cytometry was performed using Accuri C6 Flow Cytometer and analyzed on FlowJo v10.0.7. Values are mean ± S.E.M. (n = 6). c Extracellular H₂O₂ levels of explanted fibroblasts from Pparb/d^fl/fl and FSPCre-Pparb/d^−/− colons detected by Amplex Red Assay as described in legend of Fig. 3f. Values are mean ± S.E.M. (n = 6). d, e Intracellular H₂O₂ levels of explanted fibroblasts from Pparb/d^fl/fl and FSPCre-Pparb/d^−/− colons detected by CellROX. Cells were first positively gated for fibroblast marker (CD140a or PDGRA) and the CellROX fluroscence readings were taken from the CD140a + gated cells. Values are mean ± S.E.M. (n = 4). fRelative fold change in mRNA levels of 13 genes associated with oxidative stress response in explanted fibroblasts from Pparb/d^fl/fl and FSPCre-Pparb/d^−/− colons. Total RNA was extracted, and RT-qPCR was performed as described in legends of Fig. 1d, e. were Ribosomal 18S rRNA served as a housekeeping gene. Data are represented as mean ± S.E.M. from 4 independent experiments. * p < 0.05, ** p < 0.01. g, h, k, n Relative fold change in mRNA levels of 13 genes associated with oxidative stress response in HCT116 (g, k, n) and HT29 cells (h) cultured in conditioned medium of fibroblasts from Pparb/d^fl/fl and FSPCre-Pparb/d^−/− (g, h), or co-cultured with BJ-1 (k) and CCD18Co cells (n) whose endogenous PPARβ/δ was suppressed by siRNA. Cells transfected with siRNA of PPARβ/δ and Scrambled were denoted by superscript. Data are represented as mean ± S.E.M. from four independent experiments. * p < 0.05, ** p < 0.01. i, l, o Intracellular oxidative stress levels in HCT116 cells (i, l, o) either cultured in condition medium of fibroblasts from Pparb/d^fl/fl and FSPCre-Pparb/d^−/− (i) or co-cultured with BJ-1 (l) and CCD18Co cells (o) whose endogenous PPARβ/δ was suppressed by siRNA. Cells transfected with Scrambled siRNA served as cognate controls. Intracellular ROS level of HCT116 cells cultured in conditioned medium from or co-cultured with PPARβ/δ-deficient fibroblasts as determined by CM-H₂DCFDA assay. Values are mean ± S.E.M. (n = 4). j, m Relative fold change in PPARβ/δ mRNA expression in BJ-1 fibroblasts (j) and CCD18co myofibroblasts (m) whose endogenous PPARβ/δ was suppressed by siRNA at 48 and 72 h post transfection. Cells transfected with Scrambled siRNA served are cognate controls. 18 S rRNA served as housekeeping gene. Data are represented as mean ± S.E.M. from four independent experiments. * P < 0.05, ** P < 0.01