Extended Data Fig. 3: Genetically-expressed Cx43 is necessary for biotinylated molecules to distribute beyond infected cells.
From: Astrocytes connect specific brain regions through plastic networks
a. Primary astrocytes isolated from Slc1a3:Cre-ERT2+/+ x Gja1fl/fl x Gjb6fl/fl mice (GFAP, green) demonstrate distributed streptavidin staining and punctate HA-tag staining when infected with AAV5-GfaABC1D-Cx43:TID:HA and given biotin. b. Only HA-tag is visible when AAV is given without biotin. Without AAV (c) or with neither AAV nor biotin (d), neither HA-tag nor streptavidin are detected. e. Astrocytes induced for Cx43 and Cx30 knockout via 4-OHT are only positive for streptavidin in HA-tag positive cells in the AAV and biotin condition. As in b–d, only HA-tag is visible when AAV is given without biotin (f). Without AAV (g) or with neither AAV nor biotin (h), neither HA-tag nor streptavidin are detected. i. Primary mouse astrocytes isolated from uninduced cKO mice were incubated with a range of MOIs to estimate the viral load necessary for ~10% of astrocytes to express Cx43-TID. Across each MOI tested other than 1 ×103, about 80% of astrocytes were positive for biotinylated fluxed molecules; for 1 ×103, ~60% of astrocytes were streptavidin positive. j. 85.2 ± 1.6% of cells from the control AAV+ and Biotin+ condition (MOI = 1 ×104) were positive for streptavidin; the other 3 control conditions contained no detectable streptavidin signal. For cKO astrocytes induced with 4-OHT, 15.7 ± 2.6% of cells from the AAV+ and Biotin+ condition were positive for streptavidin; the other 3 cKO condition contained no detectable streptavidin signal. k. ~ 10% of cells were positive for HA-tag in all AAV+ conditions (control ++ 10.3 ± 1.2%; control +- 10.4 ± 1.1%; cKO ++ 10.3 ± 1.7%; cKO +- 10.4 ± 0.8%); no AAV- condition contained detectable HA-tag immunopositivity. n = 3 isolations for both j and k. Statistical significance (p values for all comparisons < 0.0001) determined via one-way ANOVA followed by Tukey’s multiple comparisons test (α = 0.05). Values are mean ± standard deviation.
