Skip to main content
Fig. 5 | Journal of Neuroinflammation

Fig. 5

From: Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis

Fig. 5

PC12 cells expressing α-SNC activate microglia cells to produce oxidants. a PC12 cells expressing α-SNC or α-SNC/p25α were co-cultured with unstimulated or LPS (100 ng/ml)-primed Ra2 microglia expressing the H2O2 biosensor HYPER3 overnight prior to the measurement of HYPER3 fluorescence by flow cytometry. Data are presented as mean relative fluorescent units (RFLU) +SEM. Conditioned medium was collected for WB analyses of α-SNC (inset). Data and representative image from three independent experiments. b Quantification of α-SNC in conditioned media from a. Data are presented as mean + SEM normalized to α-SNC-expressing PC12 cells (α-SNC) (*p < 0.05, N = 3). c Conditioned media were collected from PC12 cells expressing α-SNC or α-SNC/p25α after 2 days on transgene expression and transferred to Ra2 microglia expressing HYPER3. After a 2-h incubation, HYPER3 fluorescence was measured for 60 min without (Basal) or with 100 ng/ml PMA to activate NADPH oxidase. Data from a single experiment performed in triplicate wells is presented as a 485/420 nm ratio of Hyper3 fluorescence (RFLU). d Same as c but microglia were exposed to LPS (100 ng/ml) overnight prior to challenge with PC12-conditioned medium. 100 mM DTT or 100 μM H2O2 was added to obtain emission of the fully reduced/oxidized HYPER3 biosensor probe for comparison. Data from a single experiment performed in triplicate wells is presented as a 485/420 nm ratio of HYPER3 fluorescence (RFLU). e Bar graphs showing quantification of maximal oxidant response from (cd). Data are presented as mean + SEM percent normalized to control cells (*p < 0.05, N = 3). f PC12 neurons expressing α-SNC were co-cultured with Ra2 microglia in the absence (top row, control) or presence of 100 ng/ml LPS (6 h, bottom row). Immunofluorescence stain shows the distribution of α-SNC, CD11b, and TLR2 as indicated. Right side panels show outlined cell in presence of LPS at higher magnification to demonstrate colocalization of α-SNC with CD11b and in particular TLR2. Bar, 10 μm

Back to article page