Fig. 4

Activated Müller cells cause microglia activation via ATP/P2X7R pathway. a Representative immunoblots showing the changes in TSPO and P2X7R protein expression in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the P2X7R blocker BBG. Müller cells were pre-activated by incubating with DHPG for 72 h. BBG (10 μM) was added to culture medium of microglia 2 h before the activated Müller cells were added to the Transwell system. b, c A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts (b for TSPO; c for P2X7R). d Representative western blotting results showing the changes in protein levels of TSPO and P2X7R in cultured microglia which were co-cultured with normal or pre-activated Müller cells in the absence or presence of the Cx43 blocker Gap62. Gap26 (200 μM) was added to culture medium of Müller cells. e, f A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of treatment is shown in the bar charts (e for TSPO; f for P2X7R). All the data are normalized to empty transwell group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. empty transwell group using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (b, c, e, f); ^#P < 0.05 and ^###P < 0.001 using unpaired two-tailed Student’s t test against the activated Müller cells group (b, c, e, f). All in vitro experiments: n = 3 biological replicates × 3 technical replicates