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. 2018 Feb 22;3(4):e97919. doi: 10.1172/jci.insight.97919

Figure 6. αA-crystallin, secreted by Müller glial cells, protects retinal neurons against serum deprivation-induced cell death.

Figure 6

(A) Representative Western blot images of whole-cell lysate and the media of Müller glial cells transfected with a vector overexpressing αA-crystallin and corresponding graphic representation. The serum-free culture media were recovered after 24 hours, concentrated, and treated or not with proteases (proteinase K) and permeabilizing agent Triton X-100 before the Western blot. (B and C) Protective effect of secreted αA-crystallins on retinal neuronal survival. Retinal neurons were treated for 4 hours with serum-free concentrated media, recovered from Müller cells, transfected with the empty vector (EV) or αA-crystallin WT (αA-WT), the phosphomimetic mutant (αA-T148D), or the nonphosphorylatable mutant (αA-T148A) of αA-crystallin on threonine 148 (B) or transfected with the EV and αA-T148D (C). (B) Representative Western blot images for αA-crystallin on the neuronal cell lysates, processed for the DNA fragmentation ELISA after 4-hour incubation with the different crystallin-enriched media (lysate) and Western blot of the concentrated media from transfected Müller cells (media). Relative amounts are normalized by volume. The graph depicts the DNA fragmentation ELISA data. (C) Representative Western blot images for αA-crystallin on the neuronal cell lysates, processed for the DNA fragmentation ELISA after 4 hours of incubation with αA-T148D crystallin-enriched media (lysate) with (+Ab) or without (-Ab) addition of an αA-crystallin–neutralizing antibody before the 4-hour incubation or neurons incubated with media from EV-transfected Müller cells and Western blot of the corresponding concentrated media from transfected Müller cells (media). The graph depicts the DNA fragmentation ELISA data (bottom panel). Each endpoint was measured on a minimum of 3 technical replicates in at least 2 independent experiments. ****P ≤ 0.0001, significantly different from untreated control cells; ##P ≤ 0.01; ####P ≤ 0.0001, significantly different from 4h EV media. Statistical analysis was by unpaired 2-tailed t test (A) and 1-way ANOVA followed by Student-Newman-Keuls test (B and C).