FIGURE 4.

IKKα, IKKβ, and p100/p52 are essential for the directionality of cell movement toward HMGB1. Cells were exposed to 0–30 ng/ml HMGB1 gradients in μ-Slides for 1 h, and their movement was recorded by time-lapse microscopy. Red and black lines are cell tracks moving toward the HMGB1 gradient or moving in other directions, respectively. Directional tracks are defined as those with ending points closer to the higher HMGB1 concentration in the gradient compared with their starting points, whereas nondirectional tracks are the opposite, and indeterminate tracks starting and ending at the same distance from the HMGB1 gradient (i.e., moving laterally) are also considered nondirectional tracks. The effects of IKKα, IKKβ, or p100/p52 ablation on the percentage of tracks moving toward the HMGB1 gradient or in other irrelevant directions are shown in bar graphs adjacent to cell tracks plots. The differences in the numbers of directional versus nondirectional tracks between WT and each mutant MEF cell population were always statistically significant (χ² test with p < 0.01 in all comparisons). A and B, IKKβ^f/f:CreER^T1 MEFs versus the same cell population pre-exposed to 4-OHT for 36 h to ablate IKKβ expression. C and D, IKKα^f/f:CreER^T2 MEFs versus their 4-OHT–treated IKKα-ablated counterparts. E and F, Immortalized WT versus p100/p52^−/− MEFs. Experiments were repeated two to four times with comparable results. A descriptive view of μ-Slides and how cell movement was followed by time-lapse microscopy is also provided in Supplemental Fig. 4.