Fig. 2

C3a activates astrocytes, promoting MB cell proliferation in vitro. a–d Purified primary MB cells were treated with C3a at the indicated concentration for 48 h in vitro. Immunostaining of Ki67 in red color (a and b) or cleaved caspase 3 (CC3, c and d) in green color was performed to determine the proliferation and apoptosis of MB cells. DAPI-counterstained cell nuclei are shown in blue color. Quantifications of the percentages of Ki67 + cells (b) and CC3 + cells (d) were plotted according to a and c, respectively. e–f Primary astrocytes were stimulated with C3a at the indicated concentration for 48 h in vitro. Immunostaining of GFAP (green) was performed to examine astrocyte activation (e), and the percentage of GFAP + cells was quantified (f). Astrocytes were also harvested after C3a administration for GFAP expression assessment by western blotting at the protein level (g) and qPCR at the mRNA level (h). GAPDH served as a protein sample loading control. **p < 0.01, ***p < 0.001 vs. (0 nM C3a) group in b, d, f and h. ns: not significant. i, j Astrocytes were pretreated with C3a at the indicated concentration for 24 h and washed thoroughly before being cocultured with MB cells at a 1:10 ratio for another 48 h. Then, the cells were immunostained with Ki67 and GFAP (i), and the percentage of Ki67 + /GFAP− cells was quantified (j). **p < 0.01, ***p < 0.001 vs. (MB alone) group in j. Images are representative, and values are expressed as the mean ± SEM from at least three independent experiments