Journal: Frontiers in Endocrinology

Article Title: Loss of the PDLIM2 protein during chronic colitis promotes inflammation, impaired epithelium recovery, alterations to the microbiome and oxidative stress

doi: 10.3389/fendo.2025.1720162

Figure Lengend Snippet: Persistent metabolic stress and impaired anti-oxidant buffering in Caco-2 cells with PDLIM2 suppressed. (A) Protein expression of PDLIM2 and Glut-1 in shScrm and shDPLIM2 Caco-2 cells that were cultured in complete medium. (B) Gene expression of HK-2 and BACH1 determined by qPCR using cDNA generated from mRNA isolated from shScrm and ShPDLIM2 Caco-2 cells. (C) Western blots showing p-AMPK (T172), AMPK, and Tubulin as a loading control in cell lysates derived from shScrm or shPDLIM2 Caco-2 cells that were cultured in either high or low glucose-containing medium for 48hrs. (D) Mitochondrial membrane potential was assessed by flow cytometry in adherent shScrm and shPDLIM2 cells by incubation in medium containing 500nM TMRE dye for 20 minutes, before detachment and analysis. In addition, Carbonyl cyanide m-chlorophenyl hydrazone (20µM CCCP) was added 20 minutes as a control for mitochondrial uncoupling (bottom two histograms). Data are presented as mean ± SEM of geomean fluorescence from three independent experiments. (E) Adherent cells were incubated with 10µM of H 2 DCFDA for 30 minutes in the dark to quantify hydrogen peroxide species, 30 minutes with 5µM of CellROX to quantify superoxide species, or 10 minutes with 500nM MitoSOX to quantify mitochondrial-specific ROS. Cells were photographed using 100x magnification with a fluorescence microscope. The mean fluorescence intensity was quantified per image field using image J for all probes and the scale bar in representative images correspond to 50µm. (F) Left bar graph: RT-qPCR analysis of NFE2L2 , NQO1 , SOD2 , and GPX1 gene expression in shScrm and ShPDlim2 cells following culture for 48 hours. A representative western blot showing PDLIM2, catalase, HO-1, SOD2, and Tom20 protein expression in these cells. Right bar graph shows protein expression normalized against levels of Catalase, SOD2 or HO- in ShScrm cells, set to 1. (G) ShScrm and shPDLIM2 Caco-2 cells were cultured in the presence of 10µM MG132 for 4 hours and then stimulated with 5µg/mL fibronectin for 15 minutes prior to cell lysis and western blotting to quantify expression levels of NRF2, FAK, p-FAK (Y397), and PDLIM2. Bar graphs on right show fold change in NRF-2 and p-FAK expression, respectively. (H) Cells were incubated with CellROX orange for 30 minutes prior to stimulation with 5µg/mL fibronectin for 0, 15, 30, 60, or 120-minutes and photographed using 100x magnification using a fluorescence microscope. The bar graph shoes the mean ± SEM of CellROX staining from three independent experiments. All bar graphs depict the mean ± SEM of three independent experiments. P-values were calculated using the student’s t-test and where * = P < 0.05, ** = P < 0.01, and *** = P < 0.001.

Article Snippet: Blots were probed using the following antibodies: Rabbit anti-NRF2 (#12721), Rabbit anti-FAK (Y397) (#8556), rabbit anti-HO-1 (5061), rabbit anti-catalase (#14097), rabbit anti-AMPK (#2532), rabbit anti-p-AMPK (T172) (#2535), rabbit anti-GAPDH (2188), mouse anti-ubiquitin (#3936), and rabbit anti-β-catenin (#8480) were acquired from Cell Signaling.

Techniques: Expressing, Cell Culture, Gene Expression, Generated, Isolation, Western Blot, Control, Derivative Assay, Membrane, Flow Cytometry, Incubation, Fluorescence, Microscopy, Quantitative RT-PCR, Lysis, Staining