Fig. 2

AHR modulation alters ALDH1 activity in Hs578T cells. (a) Wildtype or doxycycline (dox)-inducible shAhr-transduced Hs578T cells were transfected with CMV-green control plasmid and AHR-driven pGudLuc reporter and treated for 48 hours with 10 μM AHR inhibitor CH223191 or with doxycycline (1.5 μg/mL) to induce the shAhr. Ahr mRNA was quantified by RT-qPCR and normalized to Gapdh mRNA expression. pGudLuc activity was assayed by luminescence and normalized to CMV-green expression. Values were normalized to Ahr or pGudLuc levels in untreated Hs578T cells. Data are presented as mean ± standard error. Left panel n = 6, middle panel n = 3, right panel n = 6. Asterisks indicate a significant decrease in the mRNA fold-change or reporter activity, *P <0.01, **P <0.001, ***P <0.0001. (b) Representative flow cytometry plots of ALDEFLUOR™ staining of wildtype Hs578T cells or dox-inducible shAhr-transduced Hs578T cells treated for 48 hours are presented. Dox-inducible shAhr transduced Hs578T cells were treated for 48 hours with dox. Hs578T wildtype cells were treated for 48 hours with vehicle, 10 μM CH223191, 1 μM β-NF, 0.5 μM FICZ, 1 nM TCDD, or 1 μM DMBA. Regions representing ALDH^high cells were drawn based on the signal generated in the presence of DEAB. (c) Hs578T cells were treated as in (b) and assayed for the percentage of ALDH^high cells. Data were normalized to results obtained with naïve cells (mean baseline = 4.7 % ALDH^high cells) and presented as mean fold-change from naive ± standard error. Number of experiments by condition: shAHR-dox n = 10, shAHR + dox n = 10, DMSO n = 24, CH223191 n = 9, FICZ n = 16, FICZ + CH223191 n = 5, β-NF n = 5, β-NF + CH223191 n = 5, TCDD n = 10, TCDD + CH223191 n = 3, DMBA n = 10, and DMBA + CH223191 n = 4. Asterisks indicate a significant decrease in the percentage of ALDH^high cells, *P <0.05, **P <0.001, ***P <0.0005. A cross indicates a significant increase in ALDH^high cells, ⁺ P <0.05, ⁺⁺ P <0.01, ⁺⁺⁺ P <0.005. (d) Depicted are flow cytometry dotplots of dox-inducible shAhr-transduced Hs578T cells without dox (red dots) versus with dox (black dots), wildtype Hs578T cells treated with vehicle (red dots) versus CH223191 (black dots), or wildtype Hs578T cells treated with vehicle (red dots) versus FICZ (black dots). Data are representative plots of independent experiments of the data presented in (c)