Fig 1: Effects of pioglitazone treatment on THT-HU1 (human telomerase reverse transcriptase-immortalized human urothelial cells). Cells were incubated in 25μM of pioglitazone-containing Dulbecco’s modified Eagle’s medium for 72 hours. Whole-cell lysates were immunoblotted with various antibodies as indicated. (A) Immunoblot analyses were performed to validate the expression levels of identified differentially expressed proteins. The protein expression levels of MYH3 and ACTG2 significantly decreased with pioglitazone treatment (PIO), compared to controls (Ctrl). (B) Immunoblot analysis compared several epithelial-mesenchymal transition markers, including Slug, Snail, N-cadherin, β-catenin, and E-cadherin, in pioglitazone treated (PIO) and untreated (Ctrl) cells. (C) Cell junction markers (ZO-1, ZO-2, and CD2-associated protein) were also measured by immunoblot analysis. As the protein loading control, levels of β-actin were shown. (D) Levels of mitochondrial oxidative phosphorylation (OXPHOS) markers were compared between Ctrl and PIO groups. No significant changes were observed in response to pioglitazone treatment. (E) Western blot analysis of phospho-NF-κB (P-NFKB), phospho-Erk/MAPK (P-Erk/MAPK), phospho-HER2/ErbB2 (P-HER2/ErbB2), phospho-p21 activated kinase 1 (P-PAK1), and phospho-glycogen synthase kinase-3β (P-GSK3β) were performed. β-actin was used as an internal control. (F) Reactive oxygen species production was compared between Ctrl and PIO groups. NS, nonsignificant. (G) Cell proliferation was quantified by trypan blue staining. **P < 0.005. (H) Dose- and time-dependent cell growth rates were measured in an independent set of experiments. *P < 0.01. **P < 0.005. NS, nonsignificant. ATP5A, ATP synthase subunit alpha; UQCRC2, ubiquinol-cytochrome C reductase core protein 2; MTCO1, mitochondrially encoded cytochrome C oxidase I; SDHB, succinate dehydrogenase; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8.
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