Effects of MW151 on suppression of diffuse brain injury-induced IL-1β in the cortex.

<p><b>(A)</b> Overview of experimental design for dual administration, dose response experiment. <b>(B)</b> IL-1β was increased in the mFPI + veh group compared to sham + veh, and MW151 suppressed the injury-induced IL-1β increase at the three doses tested (F_4,39 = 5.4895; p = 0.0013) (n = 8 sham + veh; n = 12 mFPI + veh; n = 6 mFPI + MW151 0.5mg/kg; n = 6 mFPI + MW151 1.5mg/kg; n = 12 mFPI + MW151 5mg/kg). <b>(C)</b> Overview of experimental design for single administration, single dose experiment. (<b>D</b>) IL-1β was increased in the mFPI + veh group compared to sham + veh, and MW151 suppressed the injury-induced IL-1β increase compared to mFPI + veh (F_2,14 = 3.8882; p = 0.0499) (n = 3 sham + veh; n = 7 mFPI + veh; n = 5 mFPI + MW151 5mg/kg). ^#p<0.001 compared to sham + veh. *p<0.05, **p<0.001 compared to mFPI + veh. (mFPI = midline fluid percussion injury; veh = vehicle).</p

Effects of MW151 on pSTAT3.

<p><b>(A)</b> Overview of <i>in vivo</i> TBI experimental design. (<b>B</b>) Representative example of pSTAT3 immunohistochemistry (IHC) in mFPI + veh -treated mice. Box indicates region shown at higher magnification in (<b>C</b>). Brown DAB staining is pSTAT3. Blue-green staining is a Methyl green counter stain. <b>(D)</b> Digital neuropathological quantification of pSTAT3⁺ nuclei in the cortex was done using the Aperio ScanScope and nuclear algorithm (n = 4 sham + veh, n = 9 mFPI + veh, n = 10 mFPI + MW151) (F_2,22 = 7.5286; p = 0.0037). *p<0.05, **p<0.001 compared to mFPI + veh. (mFPI = midline fluid percussion injury; veh = vehicle). <b>(E)</b> BV-2 cells were treated with veh or MW151 and stimulated with IFNγ (10μg/ml) for 60min, then cell lysates were harvested for western blot analysis. The data presented is a representative experiment (n = 2–3 samples per group), with the experiment replicated 3 times. (*p<0.05, **p<0.01 compared to IFNγ + veh). <b>(F)</b> BV-2 cells were treated with veh or MW151 and stimulated with IL-6 (1ng/ml) for 60min, then cell lysates were harvested for ELISA. The data presented is a representative experiment (n = 4–6 samples per group), with the experiment replicated 4 times. (***p<0.0001 compared to IL-6 + veh).</p

No effects of MW151 on BV-2 microglia cell engulfment of pH sensitive <i>E</i>. <i>coli</i> bioparticles.

<p><b>(A)</b> The pHrodo dye is non-fluorescent at neutral pH, but acidification, presumably in the cell phagosome, causes the dye to fluoresce in the red spectrum. Over the first 3h after adding the pHrodo-labeled bioparticles, the average mean intensity of the red calibrated unit (RCU) increased, but after 3h the RCU intensity plateaued. <b>(B)</b> At 3h, near the end of the linear phase of increasing RCU, the effect of treatment with vehicle control (saline), MW151 (7.5, 15, or 30μM), or cytochalasin D (cytD, 1 μM) was quantified. The graph represents the average of three independent experiments (mean ± SEM, n = 3), each experiment carried out in 4 replicates for each treatment. **p<0.05 compared to saline vehicle. <b>(C)</b> Representative photographs of BV-2 cells treated with saline, 30μM MW151 or 1μM CytD treatment at 0, 3, and 6 hrs after the addition of the bioparticles. Images and data obtained using Incucyte Zoom at 20x objective.</p

No effects of MW151 on BV-2 microglia cell migration into scratch wound.

<p><b>(A)</b> Representative graph of the rate of BV-2 cell migration into wound area, as determined by the percent confluency in the area left nearly devoid of cells after the scratch wound, and plotted as percent wound closure. <b>(B)</b> At 12h, during the linear phase of the wound closure, the effect of vehicle control (saline), MW151 (7.5, 15, or 30μM), or cytochalasin D (CytD, 1 μM) was quantified, as in (A), and plotted as percent of saline vehicle. The graph represents the average of three independent experiments (mean ± SEM, n = 3), each experiment carried out in 8 replicates for each treatment. **p<0.01 compared to saline vehicle. <b>(D)</b> Representative photographs of BV-2 cells migrating into scratch wound area with saline, 30μM MW151 or 1μM CytD treatment at 0, 6, 12, and 24 hrs after initial scratch. Blue lines indicate initial scratch wound area. Pink is wound area at each time point, calculated by Incucyte Zoom software. Images and data obtained using Incucyte Zoom at 10x objective.</p

No effects of MW151 on BV-2 microglia cell growth curves.

<p><b>(A)</b> Representative example of BV-2 cell growth curve over the first 60h after plating in a 96 well plate at 5,000 cells/well. <b>(B)</b> BV-2 cells were treated at 6h after plating, with vehicle control (saline), MW151 (7.5, 15, or 30μM), or cytochalasin D (CytD, 1 μM). Each experiment was carried out in 8 replicates, with graph summarizing 3 independent experiments (mean ± SEM, n = 3). **p<0.01 compared to saline. <b>C)</b> Representative photographs of BV-2 cells treated with saline, 30μM MW151, or 1μM CytD at 0, 12, 24, 36 and 48 hrs after drug treatment. All imaging was done using Incucyte Zoom at 10x objective.</p