Fig. 8
AHR modulation affects markers associated with BCSLC in SUM149 cells. (a) Ahr, Cyp1b1 and Aldh1a1 mRNA expression in sorted ALDHhigh and ALDHlow SUM149 cells was quantified by RT-qPCR. Data from three independent experiments were analyzed using the Pfaffl method, normalized to the Gapdh signal, and presented as mean fold-change from ALDHlow ± standard error. Asterisks indicate a significant increase in the mRNA fold-change, *P <0.05, **P <0.01, ***P <0.005. (b) Wildtype or dox-inducible shAhr-transduced SUM149 cells were transfected, treated and quantified as indicated in Fig. 2a. Data are presented as mean ± standard error. Left panel n = 5, center panel n = 4, 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. (c) Representative flow cytometry plots of ALDEFLUOR™ staining of dox-inducible shAhr-transduced SUM149 cells cultured, treated, and depicted as described in Fig. 2b. (d) Dox-inducible shAhr-transduced or wildtype SUM149 cells were treated, stained, and quantified as in Fig. 2c. Data were normalized to results obtained with naïve cells (mean baseline =10.4 % ALDHhigh cells) and presented as mean fold-change from naive ± standard error. shAHR-dox n = 4, shAHR + dox n = 4, DMSO n = 12, CH223191 n = 5, FICZ n = 6, FICZ + CH223191 n = 5, β-NF n = 4, β-NF + CH223191 n = 4, TCDD n = 8, TCDD + CH223191 n = 3, DMBA n = 5, and DMBA + CH223191 n = 3. Asterisks indicate a significant decrease in the percentage of ALDHhigh cells, *P <0.05, **P <0.01, ***P <0.0005. A cross indicates a significant increase in ALDHhigh cells, + P <0.01, ++ P <0.005. (e) Representative images of primary (day 8) and secondary (day 16) tumorspheres after SUM149 cells were treated for 48 hours with vehicle or 10 μM CH223191. The percent viable cells are included on each image. Data are representative of four independent experiments. (f) SUM149 cells were treated as in (e) and tumorsphere formation was quantified. Data from four experiments were normalized to results obtained with naïve cells and presented as mean fold-change from naive ± standard error. Asterisks indicate a significant decrease in the percentage of tumorspheres, *P <0.05, **P <0.01