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. 2020 Jan 3;40(2):e00307-19. doi: 10.1128/MCB.00307-19

FIG 7.

FIG 7

ALDH1A1 is regulated by SOX9 at the transcriptional level. (A) SOX9 overexpression induced by doxycycline (Dox) for the indicated length of time stimulates ALDH1A1 expression. The blots shown are representative of those from two independent experiments. (B) Schematic image of the ALDH1A1 promoter and locations of the primer sets used for the assay whose results are presented in panel C. (C) SOX9 binding is enriched at the ALDH1A1 promoter. SOX9 ChIP results were analyzed by qPCR with primers specific to the indicated DNA regions. IgG was used as a negative immunoprecipitation control. Primers to COL2A1 served as negative and positive controls for ChIP. The data are presented as the mean ± SD. P values were determined by two-way ANOVA with Bonferroni posttests. **, P < 0.01; ns, not significant. (D) SOX9 induces ALDH1A1 luciferase reporter activity in HEK293T cells. Cells were transfected with the indicated constructs, and luciferase activity was measured at 48 h posttransfection. Bars are the mean ± SEM. The indicated P value determined by an unpaired two-tailed t test. Luc/Ren, firefly luciferase/Renilla luciferase ratio. (E) Luciferase reporter assay with the ALDH1A1 promoter. H460 cells stably expressing the empty vector or SOX9 were transfected with the ALDH1A1 luciferase reporter plasmid and the empty luciferase vector. Luciferase activity was analyzed at 48 h posttransfection and normalized to the Renilla luciferase signal. ***, P = 0.028 by an unpaired two-tailed t test and P < 0.0001 by one-way ANOVA with Bonferroni’s multiple-comparison test. Bars are the mean ± SEM. The results are representative of those from two independent experiments. (F) Schematic representation of the truncated (trunc) ALDH1A1 luciferase reporters (left) and luciferase reporter assay with the full-length and truncated ALDH1A1 promoters, performed as described in the legend to panel E (right). Bars are the mean ± SEM. P values were determined by one-way ANOVA with Bonferroni’s multiple-comparison test. **, P < 0.01 versus SOX9 wild-type (WT) ALDH; ns, not significant versus SOX9 wild-type ALDH.