Fig. 7. EVI1 variants are regulated in an autophagy-independent and β-catenin-independent manner in chronic iron-exposed FT194 cells.

a Untreated and FAC-treated FT194 cells (p = 23, day 78) were treated with 10 μM or 25 μM HCQ for 18 h. Lysates were collected and subjected to western blot analyses with the indicated antibodies. The dotted line indicates the same samples run on 10% (top) or 8% (bottom) SDS-PAGE gels, and data are representative of four independent replicates. b Untreated and FAC-treated FT194 cells (p = 28, day 99) were treated with 25 μM HCQ and/or 5 μM MG132 for 18 h, and western blot analyses were completed with the indicated antibodies. The dotted line indicates the same samples run on 10% (top) or 8% (bottom) SDS-PAGE gels, and data are representative of three independent replicates. RNA was isolated from untreated and FAC-treated FT194 cells following (c; p = 26, day 92) 18 h treatment with 25 μM HCQ, or (d; p = 39, day 136) 18 h treatment with 100 nM Act D, to assess the indicated EVI1 splice variants via real-time PCR. Data represent three independent experiments. Western blot analyses were completed with the indicated antibodies following siRNA knockdown of EVI1 (siB, e; p = 32, day 113), β-catenin (f; p = 36, day 124), and BMI1 (g; p = 29–30, day 102–104). Data are representative of three independent experiments. RNA was isolated from untreated and FAC-treated FT194 cells following transfection with siRNAs targeting EVI1, siB (h; p = 27, day 86), β-catenin (i; p = 24, day 81), and BMI1 (j; p = 31, day 109), to assess hTERT mRNA expression (relative to respective control nontargeting siRNA in both untreated and FAC-treated cells) via real-time PCR. Data represent three independent replicates. k FAC-treated FT194 cells (p = 34, day 120) were maintained in the absence of 250 nM FAC for 4 days, as indicated in the “Materials and methods” section. Western blot analyses were completed with the indicated antibodies and data are representative of three independent experiments