PMC

A. The levels of activated caspase-1 and the mature IL-1β proteins observed 3 h after reperfusion in WT, Panx1 KO (KO) and Panx1 CKO (CKO) retinas. B. Quantification of the changes in band intensities from western blots in figure A (n = 3, ** p<0.05). C. The IHC analysis showing active IL-1β (17 and 20 kDa isoforms, arrowheads) in the cells of the inner retinal layer (INL) after IR challenge. Caspase1-specific (red) and IL-1β-specific (green) staining co-localized with RGCs and other cells in the ganglion cell layer (GCL), IL-1β staining also labeled cells in the INL. DAPI staining for nuclei is blue. Scale bar, 50 µm.

A. Representative micrographs of retinas from experimental (post-IR) and control sham treated (sham) eyes from WT, Panx1 CKO (CKO) and Panx1 KO (KO) animals challenged by retinal IR. RGCs are labeled with Neuronal ClassIII β-Tubulin antibodies for counting. Scale bar, 50 µm. B. Neuronal survival was calculated as the percentage of cells in experimental vs. fellow sham-operated eyes (mean±SD, n = 8); RGC survival rates were measured at 7 and 14 days after IR using direct cell count in retinal flat-mounts. C. RGC Survival and the rates of apoptotic and necrotic cells in the OGD-challenged primary RGCs cultures from Panx1 KO and WT retinas 24 h after OGD. Annexin V/Propidium Iodide (PI) co-labeling was utilized to analyze cell death.

A. RT-PCR analysis of retinal and brain homogenates from wild type (WT), Panx1 CKO (CKO) and Panx1 KO (KO) mice using primers for the floxed Exon 4 (287 bp band, arrow). B. Western blot analysis of total retina homogenates probed with rabbit anti-Panx1 CT-395(Px-34) antibodies shows a 47 kDA band (arrow) corresponding to Panx1. C. Quantitative RT-PCR analysis of relative abundance of Panx1 transcripts in total retinal extracts from the animals of all three genotypes. D. Relative abundance of Panx1 and Panx2 transcripts in total extracts from WT, CKO and KO retinas. The values were normalized to the levels of Actb (β-actin), with the data presented as mean±SD. Double asterisk indicates P<0.01. E. Confocal micrographs of retinal sections from WT and KO mice probed with anti-Panx1 CT-395 antibodies (red) and co-labeled with RGCs marker Neuronal Class III β-Tubulin (green), and nuclei marker DAPI (blue). RGC somatae with Panx1-specific labeling are indicated by arrows. Scale bar, 25 µm. F. Relative abundance of Panx1, Panx2 and Cx36 transcripts the WT, CKO and KO retinas. Transcript abundances were normalized to the levels of Actb; data are presented as mean±SD. Double asterisk indicates P<0.01.

A Representative confocal images of the ganglion cell layer in whole retina flat mounts preloaded with calcein-488. Images were taken after 30 min normoxic or OGD treatment in a hypoxic chamber. Scale bar, 25 µm. Graph shows total calcein fluorescence intensity (mean ± SE, n = 4) after treatment of WT or Panx1 KO retinal explants. The reduction of fluorescence after OGD indicates the dye leakage in WT. B. Real-time confocal images of cultured primary RGCs loaded with calcein-488. Shown are representative series of three independent experiments. C. The dynamics of calcein-488 dye efflux calculated as the change in fluorescence intensity recorded from at least 50 individual cells in three independent experiments. D Intracellular Ca²⁺ changes were measured in cultured primary RGCs loaded with ratiometric fura-2 dye. OGD was induced at the “0” time point by replacing culture media with oxygen-depleted glucose-free media supplemented with 10 mM 2-deoxyglucose and 1 mM KCN. Fluorescence measurements were taken at 510 nm with excitations at 340 and 380 nm in real time with 20 sec intervals. Traces represent the average values (mean ± SE) of the 340/380 ratios obtained from a minimum of 30 individual RGC cells in a minimum of three biological repeats. In WT cells Panx1 blockade was induced by application of 10 µM CBX 20 minutes prior to recording.