Figure 4
Multiple HT-CM-SPR screening hit compounds alleviate αSyn mediated inhibition of phagocytosis in H4-Neuroglioma cells. H4 neuroglioma cells over-expressing αSyn from a tetracycline-inducible promoter were cultured for 24 hours with compound in the absence or presence of αSyn induced by tetracycline. After 24 hours of induction cells were (a) fed 4 micron beads for 90 minutes and a phagocytic index was measured by quantitating the amounts of engulfed beads on an imaging reader or (b) analyzed by Western blot to determine αSyn levels. (a) The phagocytic capacity was calculated by normalizing the indicated samples to the phagocytic capacity of un-induced cells not overexpressing αSyn (Tet off). Each point corresponds to a separate experiment denoting the average of 6 well replicates. Means ± SD for the combined multiple experimental averages are shown. Control compound is 484228, identified in a prior in silico screen20. n = 2 or 3 different experiments as shown. Significance was determined by ANOVA with Dunnet’s correction. *p < 0.05, ***p < 0.001, ****p < 0.0001. ns is not significant. (b) Cells were untreated or treated with tetracycline to induce αSyn and treated with DMSO or compounds and analyzed by Western blot for actin and αSyn levels. Left: Representative Western blots of merged actin and αSyn signals of individual wells treated with DMSO or compounds. Image cut as shown to remove irrelevant samples. Entire length of blot shown. Outline of full blot shown by black lines. Right: Westerns from multiple replicates were quantitated and the αSyn band intensity was normalized to that of actin. Each data point is a separate well. No compounds showed significant impact on αSyn levels. Compound structures are also shown in Supplementary Fig. S4.
