Fig. 2.

Hinokitiol allows ⁵⁹Fe³⁺ entry into HEK293 cells capable of expressing mouse 1B/− IRE DMT1. Linear regression generated a fit and 95% confidence interval (CI) for each panel. a Entry reliant on endogenous transport. b Entry reliant on C2d. c Entry reliant on hinokitiol. d Entry reliant on induced DMT1 expression. e Entry reliant on induced DMT1 expression plus C2d. f Entry reliant on induced DMT1 expression plus hinokitiol. Comparison of panels a and d reveals a × 1.3 increase in rate (P = 0.0021 by 1XANOVA) due to entry of Fe³⁺ dependent on increased expression of DMT1. This modest but significant increase can likely be attributed to the presence of a surface ferrireductase and very tight experimental control of the assay rather than Fe³⁺ imported by DMT1. Similarly, panels b and e compare entry with C2d to entry with increased DMT1 plus C2d; DMT1 provides a × 1.5 increase in rate (P = 0.001 by 1XANOVA). Again the difference is likely to be attributable to the presence of a surface ferrireductase and very tight experimental control of the assay. Comparison of panels a and b reveals no significant difference (P = 0.51 by 1XANOVA); while comparison of panels d and e also reveals no significant difference (P = 0.83 by 1XANOVA). Both comparisons confirm that C2d does not support entry of Fe³⁺ and is thus an appropriate control. The key comparisons are of the panel c (hinokitiol) to its controls—a (endogenous) or b (C2d); they reveal that hinokitiol stimulated entry by × 18.6 and × 15.2, respectively (P = 0.0001 and P = 0.0001 by 1XANOVA). (Other comparisons and corrections for multiple comparison are omitted because the focus again is only on experimentally relevant comparisons.)