Fig 2: TRPV4 regulates stretch-induced release of IL-6. E17 epithelial cells were isolated and seeded on bioflex plates coated with fibronectin. 24 hours later, cells were exposed to 20 % cyclic stretch for 48 h in the presence or absence of the vehicle DMSO, the TRPV4 agonist GSK1016790A [100 nM] or TRPV4 antagonist HC-067047 [1 μM]. Unstretched cells served as controls. Supernatants were collected and processed to assess IL-6 concentrations by ELISA, as described in methods. Values are mean ± SEM from 5 different experiments. Results were normalized to the cell lysate protein concentrations. *p < 0.05 vs control vehicle; **p < 0.01 vs stretch vehicle. Tukey-Kramer Multiple Comparisons Test. Veh = vehicle, DMSO; ag = agonist GSK1016790A; ant = antagonist HC-067047

Fig 3: Effect of mechanical stretch on IL-10 and IL-6 release into the supernatant of type II epithelial cells. A) E18 type II cells were exposed to 20% cyclic stretch for 24 hours. Supernatant was collected and measured for concentrations of IL-10 by ELISA, as described in methods. Results were normalized to the cell lysate concentration in each sample (n = 5, *P<0.02 vs control). B) Samples were processed as above, except that IL-6 release was also measured in type II cells isolated from IL-10 knockout mice (n = 3, *P<0.05 vs control wild-type; n = 3, **P<0.05 vs control IL-10 KO).

Fig 4: Release of IL-6 by stretch or TRPV4 agonist is regulated via ERK and p38 pathways. a E17 epithelial cells seeded on bioflex plates coated with fibronectin were preincubated for 30 min with the ERK pathway inhibitor U0126 [20 μM] or the p38 inhibitor SB203580 [20 μM] and then exposed to 20 % cyclic stretch for 48 h. Supernatants were collected and the concentration of IL-6 was analyzed by ELISA, as described in methods. Data are normalized to the cell lysate concentrations. N = 4; *p < 0.01 vs vehicle control; **p < 0.01 vs vehicle stretch. Tukey-Kramer Multiple Comparisons Test. b E17 epithelial cells seeded on fibronectin-coated plates were incubated for 48 h with the ERK pathway inhibitor U0126 [20 μM], the p38 inhibitor SB203580 [20 μM] or the JNK inhibitor SP600125 [20 μM], in the presence of DMSO (vehicle) or the TRPV4 agonist GSK1016790A [100 nM]. Supernatants were collected and the concentration of IL-6 was analyzed by ELISA, as described above. N = 3; *p < 0.05 vs negative vehicle; **p < 0.01 vs TRPV4 agonist. Tukey-Kramer Multiple Comparisons Test

Fig 5: Effect of PUG on the pro-inflammatory cytokine interleukin-6 (IL-6) on activated RAW264.7 macrophage cells. RAW264.7 cells were pretreated without or with LPS (1 μg/mL) for 1 h at 37 °C. Then the cells were treated with PUG (4, and 6μM) for 24 h at 37 °C. The treated cell culture media were collected and used for the IL-6 determination. The wells were read, and absorbance values were used for the graph plot. The standard deviation and error were calculated from the replicates of the samples and the Four Parameter Logistic curve (4PL) method was used to determine the IL-6 concentration. The statistical significance was ### p < 0.001 vs. untreated group; and * p < 0.05, ** p < 0.01 vs. LPS-alone treated group.