Fig. 6

The Akt-activated Nrf-2 likely reverses the SC79 inhibition of IL-8 transcription in A549 cells. a A549s were treated with flagellin (1 μg/ml) ± SC79 (2.5 μg/ml) ± L-NAME (10 μM) or D-NAME (10 μM) for 24 h and qPCR was performed. Bars are expression relative to GAPDH (2^−ΔCT; means ± SEM; n = 4 experiments).**p < 0.01 (CTL vs. flagellin) and *p < 0.05 (flagellin vs. flagellin + SC) by ANOVA and Bonferroni posttest. Raw ΔCT values were 4.79 ± 0.30 (control), 2.02 ± 0.33 (flagellin; **p < 0.01 vs control), 3.46 ± 0.17 (flagellin + SC79; *p < 0.05 vs flagellin only), 3.71 ± 0.12 (flagellin + SC79 + L-NAME), 3.69 ± 0.22 (flagellin + SC79 + D-NAME). b Brusatol (≥ 100 nM) degrades total Nrf-2 in A549 cells, and this is sufficient to increase IL-8 levels. Cells were grown and treated with brusatol at 1 nm, 100 nm, and 1 μM for 2 h before lysing the cells for Western. Bars are means ± SEM; n = 3 experiments; *p < 0.05 (CTL vs. 1 μM) by ANOVA and Bonferroni posttest. c Brusatol reduces nuclear Nrf-2 at ≥ 1 nM. A549 cells were grown and were treated with brusatol at 1 nm, 100 nm, and 1 μM for 2 h. Cell lysates were collected and centrifuged to separate nuclear fractions as described in the text. Actin shown as a loading control. Representative of 3 experiments showing complete loss of nuclear Nrf-2. d Nrf-2 inhibition alone with brusatol at higher concentrations increases IL-8 in A549 cells. A549 cells were grown and treated with 1 μM brusatol for 24 h. Cells were lysed for qPCR. Bars are expression relative to GAPDH (2^−ΔCT; means ± SEM; n = 3 experiments). *p < 0.001 (CTL vs. 1 μM brusatol) by unpaired t-test. Raw ΔCT values (control vs brusatol) were 5.96 ± 0.19 vs 0.16 ± 0.21 (p < 0.001 by unpaired t test). e ML385 alone (1 μM)) also increases IL-8 in A549 cells. Bars are expression relative to GAPDH (2^−ΔCT; means ± SEM; n = 3 experiments). p < 0.001 (CTL vs. 1 μM) by unpaired t-test. Raw ΔCT values (control vs ML385) were 5.88 ± 0.23 vs 0.26 ± 0.78 (p < 0.001 by unpaired t test) f Brusatol (1 nM) partially inhibits SC79 reduced IL-8 in A549 cells. A549 cells were grown and treated with flagellin (1 μg/ml) ± SC79 (2.5 μg/ml), and ± brusatol (1 nM) for 24 h and qPCR was performed. Bars are expression relative to GAPDH (2^−ΔCT; means ± SEM; n = 4 experiments). Significance determined by one-way ANOVA with Dunnett’s posttest; * p < 0.05 and **p < 0.01 vs control. Raw ΔCT values were 4.06 ± 0.15 (control), 2.71 ± 0.19 (flagellin; p < 0.01 vs control), 4.16 ± 0.36 (flagellin + SC79), 3.26 ± 0.29 (flagellin + SC79 + brusatol; p < 0.01 vs control), 5.55 ± 0.46 (brusatol alone). g Nrf-2 is upregulated in the nuclear fractions in response to SC79. A549 cells were treated with SC79 (10 μg/ml; DMSO only as control) for 2 h. Cell lysates were collected and centrifuged to separate nuclear fractions. Bars are means ± SEM; n = 3 experiments. **p < 0.01 (CTL vs. SC) by unpaired Student’s t-test. h Western of LaminB2 for nuclear fractions and plasma membrane Na⁺K⁺-ATPase for cytosolic fractions