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. 2022 Sep 1;17(9):e0271950. doi: 10.1371/journal.pone.0271950

Fig 1. Cigarette smoke extract (CSE) increases TNFα and EMT markers in ARPE-19 cells.

Fig 1

(A) ARPE-19 cells were treated with untreated culture medium (0% CSE) or 0.5% or 1% CSE in culture medium for 4 or 24 hours. After treatment, cells were collected and RNA was isolated. Exposure to 0.5 or 1% CSE for 4 or 24 hours induced expression of TNFα (left) and Snail (right) mRNA. mRNA levels are normalized to GAPDH mRNA levels and presented as fold change over untreated cells(0% CSE). Error bars represent standard error of the mean, with N = 3 for each point. * = p<0.05 and ** = p<0.01 compared to 0% CSE. (B) Representative cell culture images of ARPE-19 cells without CSE (0% CSE) or with 0.5 or 1% CSE at 4 and 24 hours. CSE alters the morphology of the ARPE-19 cells to become more rounded, especially at 24 hours treatment. (C) ARPE cells were treated with or without CSE as in (A) for 24 hours. Afterwards, cells were lysed and protein expression was analyzed by Western blot. CSE elevates both αSMA and SNAIL levels. (D) ARPE-19 cells were treated with 1% CSE in the presence or absence of 1 uM of the NF-κB inhibitor, BAY-11-7082 (BAY). After 24 hours, cells were harvested and protein and mRNA levels were analyzed as described above. Inhibition of NF-κB mitigated the ability of CSE to induce SNAIL and EMT marker, vimentin. (E) RNA was isolated from cells treated as in (D). BAY-11-7082 mitigated the ability of CSE to induce TNFα mRNA.