Effect of MM patient sera on HAMP promoter activity. HuH7 cells were cotransfected with WT-HAMP, mSTAT, mBRE1/2, or mAll3 promoter-firefly luciferase construct, together with pTK-RL construct, and treated with 10% serum in Optimem. After 6 hours, cells were lysed and luciferase activity was measured. (A) Treatment with MM patient sera caused a significantly higher induction of WT-HAMP promoter activity, compared with sera from healthy individuals. Results are expressed as fold induction over untreated control. Statistical significance was determined with the Mann-Whitney rank-sum test. Boxes represent median and 25-75 percentiles, and whiskers represent 10 and 90 percentiles. Circles are outliers; *P < .05, compared with normal sera. (B) Mutation of BREs abrogated induction by MM sera dramatically, while the STAT3-BS mutation had a statistically less significant effect. Results are expressed as fold induction over untreated control (construct/optimem). Bars represent mean ± SD. Statistical significance was determined with 1-way ANOVA to compare the same sera for the different mutations. To compare MM patient sera with normal sera within the same construct, the Mann-Whitney rank-sum test was used; *P < .05, compared with untreated control (construct/optimem), and †P < .05, compared with WT-HAMP promoter activity for the same sera.