Fig 2: Relationships between levels of PPARa and PIP5K1C, PIP2, PI3K, and PTEN in HT-29 cell line. (A) Spearman correlation coefficients (n = 12) for expression levels obtained with In-Cell ELISA method. PPARa expression under the same experimental condition was examined in our previous study [18]. Moderate positive correlations were found between PPARa and PIP5K1C and PPARa and PIP2, whereas PPARa and PTEN expression showed moderate negative correlation (statistical significance marked with asterisk, * p = 0.05) (B) Intensities of PIP5K1C, PIP2, and PTEN immunofluorescent staining for dependence of PPARa subcellular localization. Fluorescent intensities of PIP5K1C, PIP2 and PTEN were measured using ImageJ software. Note that intensities of fluorescence are at the same levels in cells with nucleus positive for PPARa and cells with PPARa negative nucleus (unpaired t-test, nonsignificant results, n = 30 in each group). Colocalization of PPARa and PIP5K1C (C), PIP2 (D), and PTEN (E). The cells were treated by 150 µM fenofibrate (PPARa activator) and 10 µM GW6471 (PPARa inhibitor), and controls were treated with appropriate amount of DMSO. Staining intensities of PIP5K1C, PIP2, and PTEN were independent of subcellular localization of PPARa. White arrows indicate cells with PPARa negative nuclei together with strong staining of PIP5K1C, PIP2, and PTEN. White arrow heads indicate cells with PPARa positive nuclei together with intensive staining of PIP5K1C, PIP2, and PTEN. All microphotographs are in the same magnification (400×), white line represents 10 µm.

Fig 3: Effect of PPARa activators fenofibrate (A) and WY-14643 (B) and PPARa inhibitor GW6471 (C) on expression of PIP5K1C, PIP2, PI3K, and PTEN. Relative expression of proteins of interest in comparison to control was measured by In-Cell ELISA. Results are shown as mean ± SD (n = 6). Red dotted line represents control cells: DMSO treated undifferentiated or differentiated cells (100%). HT-29 cells were differentiated by sodium butyrate, Caco2 cells were differentiated by post-confluent growth (14 days). Note the increase in PIP2 expression in both used cell lines after PPARa ligands treatment (red rectangles). Statistically significant results in comparison to control cells are marked by * p = 0.05, ** p = 0.01, *** p = 0.001, **** p = 0.0001. (D). Relationship between changes in expression of PIP5K1C and PTEN in HT-29 and Caco2 cell lines are shown as Spearman correlation coefficients. Changes in expression of these proteins after PPARa ligand treatment showed a moderate negative correlation in both cell lines. (E) Schematic summary of obtained results. Detected increase in PIP2 observed after fenofibrate, WY-14643, and GW6471 treatment could explain our previous results reported increase in villin expression under the same experimental conditions [18]. The effect of PPARa ligands is illustrated by arrows: green—increase in expression, red—decrease in expression, grey dotted—no effect, red dotted—decrease in expression or no effect (the results differ in HT-29 and Caco2 cell lines).