Skip to main content
. 2012 Nov 13;7(11):e49426. doi: 10.1371/journal.pone.0049426

Figure 5. Synergistic ENaC induction by endiandrin A is linked to an activation of p38 and ERK in HT-29/B6-GR cells.

Figure 5

HT-29/B6-GR cells were pre-incubated with the indicated MAPK inhibitors (10 µmol/L) for 1 hour before addition of dexamethasone (1 µM) and/or TNF-α (500 U/ml) and/or endiandrin A (20 µM) for 24 hours. (A) Measurement of ENaC-dependent Na+ absorption as the drop in ISC after amiloride (100 µM) and measurement of γ-ENaC-mRNA after 24 hours. GAPDH was used for normalization of mRNA expression. Data are given as means ± s.e.m., n = 8–11 and n = 5–8, ***P<0.001 compared with dexamethasone incubation, # P<0.05, ## P<0.01 and ### P<0.001 compared to dexamethasone + TNF-α 500 IU/ml, °° P<0.01 and °°° P<0.001 compared with dexamethasone + TNF-α 500 IU/ml + endiandrin A. (B) Western blot analysis of total GR protein (∼95 kDa) of HT-29/B6-GR cell lysates. Cells were pre-incubated for 1 hour with SB202190 (10 µM) before incubation with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) and/or endiandrin A (20 µM) for 3 hours. Human β-actin (∼42 kDa) served as a loading control. (C) Densitometry of SB202190 induced effects on total GR levels. Shown are means ± s.e.m., n = 7, *P<0.05 compared to dexamethasone, TNF-α 500 IU/ml and endiandrin A, # P<0.05 compared to TNF-α 500 IU/ml and endiandrin A. (D) Western blot analysis of HT-29/B6-GR cell lysates incubated with dexamethasone (1 µM) and/or TNF-α (500 IU/ml) w/wo endiandrin A (20 µM). Shown are p38 and pp38 (∼38 kDa) after 120 minutes stimulation as well as ERK and p-ERK MAPK protein (∼42/44 kDa) after 5 minutes stimulation. Human β-actin (∼42 kDa) served as a loading control.