Figure 1.
LPS-stimulated activation of microglial cells with increasing iba1-immunoreactivity in culture.
The iba1-positive cells are shown in N9 cells (A) and BV2 cells (B) with DAPI counterstaining after LPS exposure of 12h-48h. Density in mean fluorescence intensity (MFI) of iba1-immunoreactivity among different N9 and BV2 cell groups is compared (C), ANOVA: * <0.05, ** <0.01 vs control.
Figure 2.
Increase of proNGF-immunoreactivity in N9/BV2 microglial cells after LPS stimulation.
The proNGF-immunoreactivity is shown in N9 cells (A) and BV2 cells (B) with LPS exposure of 12h-48h. Density in proNGF-immunoreactivity (MFI) among different N9 and BV2 cell groups is compared (C), ANOVA: * <0.05, ** <0.01 vs control.
Figure 3.
Western blot showing LPS-induced up-regulation of proNGF expression in N9/BV2 microglial cells.
The immunoblot bands are respectively shown in N9 cells by proNGF antibody (A), BV2 cells by proNGF antibody (B), and N9 cells by NGF antibody (C). Comparison of proNGF levels is shown among distinct N9/BV2 cell groups (D). ANOVA: * <0.05, ** <0.01 vs control.
Figure 4.
Expression patterns of proBDNF in N9/BV2 cells in both control and LPS exposure groups.
Immunofluorescence shows proBDNF localization in N9 cells (A) and BV2 cells (B). Western blot shows proBDNF in N9 cells by proBDNF antibody (C), BV2 cells by proBDNF antibody (D), and N9 cells by BDNF antibody (E). Comparison of proBDNF expression levels shows no significant difference among distinct N9/BV2 cell groups (F).
Figure 5.
Expression patterns of MMP-9 in N9 and BV2 microglial cells in control and LPS exposure conditions.
Immunofluorescence for MMP-9 is representatively shown in N9 cells of control group (A), immunoblotting bands of MMP-9 are shown in both N9 and BV2 cells of control and LPS groups (B, C). Comparison of MMP-9 expression levels does not show significant difference among distinct N9 and BV2 cell groups (D).
Figure 6.
The release of proNGF from activated N9 microglial cells after LPS stimulation in cell culture.
The proNGF is shown in culture medium of N9 cells of control and LPS groups (A), and increase of proNGF level is seen in LPS group (B). Cell viability by trypan blue test in N9 cells does not show difference between control and LPS group (C). ANOVA: * <0.05, ** <0.01 vs control.
Figure 7.
Bioassay of recombinant human proNGF (rhproNGF) in the SH-SY5Y cells.
Characterization of rhproNGF is shown by western blot (A). Positive expression of p75NTR and sortilin is detected in SH-SY5Y cells in both control and LPS stimulation conditions (B). MTT shows no significant change of SH-SY5Y cell viability after proNGF treatment at different concentrations (C). Hoechst/PI staining indicates cell damage of SH-SY5Y cells resulted from proNGF treatment at 10ng/ml and 40ng/ml (D). E, Immunoblot shows a slight increase of cleaved caspase-3 expression (mainly 19kDa) following proNGF treatment (E). TUNEL assay shows appearance and increase of apoptotic cell death in SH-SY5Y cells with proNGF treatment (F).