HHQ inhibits Af preformed biofilm; comparison to PQS. (A) Dose-response study with preformed 10AF biofilm. Flat dose-response shown. “DMSO control” is same concentration of DMSO diluent as present in test article wells. Twofold dilutions of HHQ were made from 50 to 0.39 μg/mL. Bars showing results with 3.12–12.5 μg/mL were not different from the 1.56 or 25 μg/mL bars, so are not shown in order to simplify the figure. All concentrations shown were p < 0.001 compared to control except 0.78 μg/mL, which was p < 0.01. (B) through (F): molecules tested at 1.56 μg/mL. DMSO control results same as ethanol control or RPMI alone control, in every panel. (B,C), comparison with PQS. (B) HHQ and PQS inhibit strain 10AF compared to respective controls, p < 0.01 and p < 0.001, respectively. PQS more potent than HHQ, p < 0.001. (C) HHQ inhibits strain Af293 by 15%, but not significantly. PQS different from HHQ and control, p < 0.001 both comparisons. (D) Reversal of inhibition by Fe, no boosting above Fe alone at high Fe concentrations, Af strain 10AF. Fe was diluted twofold from 1000 to 0.1 μM final concentrations (these are the numbers on the x axis), in presence or absence of HHQ. Data presented as % of controls. HHQ alone was inhibitory compared to control, p < 0.001 (left two bars). At Fe 0.1 μM + HHQ (right bar), inhibition by HHQ is partially reversed; the combination still shows inhibition compared to control (p < 0.05), but less inhibition (p < 0.05) compared to HHQ alone. Fe 1–10 μM completely reverses HHQ inhibition in combination (2nd and 3rd bars from right), these are not significantly different from control, and Fe 10 μM + HHQ is p < 0.05 compared to HHQ alone. Fe alone at 10 μM (5th bar from left) begins to significantly boost Af metabolism (p < 0.05 compared to control) and higher concentrations are p < 0.001 compared to control. HHQ inhibition in combinations blunts the Fe boosting about 10-fold, since it is not until the combination of HHQ with Fe 100 μM (4th bar from the right) that there is significant boosting above control (p < 0.01; Fe 1000 μM is p < 0.001 compared to control). No combination of HHQ with high concentrations of Fe is boosted above the same concentration of Fe alone. Studies in hypoxic conditions, Figure 8E,F. In 8E, strain 10AF, HHQ and PQS inhibit to 82% and 73% of respective controls, both p < 0.001; not significantly different. In 8F, strain Af293, HHQ and PQS inhibit to 87% and 40% of respective controls, p < 0.05 and p < 0.001, respectively. HHQ and PQS effect differ by p < 0.001.