Extended Data Fig. 1: Primary immunopanned astrocyte purity and knockout induction.
From: Astrocytes connect specific brain regions through plastic networks
a. qPCR of marker gene expression for astrocytes (Gja1, Aldh1l1, Slc1a3), oligodendrocytes (Sox10, Mog), microglia (Aif1, Cd14), and neurons (Tubb3, Syp) in primary immunopanned astrocytes from rat or cKO mouse normalized to the ribosomal gene RPL19 and relative to naïve cortex from simultaneously dissected littermate controls. b. Primary cKO mouse astrocyte viability determined as the ratio of Hoechst positive, Propidium Iodine (PI) negative cells to the total number of Hoechst positive cells. Viability was significantly decreased at 4-Hydroxytamoxifen (4-OHT) concentrations of 1:2000 and above (p < 0.001). c. qPCR of Gja1(Cx43) and Gjb6(Cx30) in primary immunopanned cKO astrocytes at a 4-OHT concentrations of 1:5000, 1:4000, and 1:3000 relative to naïve cells. All 4-OHT concentrations decreased expression of both RNAs, with the largest decrease at 1:3000. d. Quantification of western blots from immunopanned cKO astrocytes in the following conditions: Naïve, Naïve + Vehicle (EtOH, 1:3000), 4-OHT 1:6000, 4-OHT 1:5000, 4-OHT 1:4000, and 4-OHT 1:3000. Blots were costained for Cx43 and Actin (example on right; all quantified blots in Extended Data Fig. 1). Cx43/Actin normalized to Naïve significantly decreased at all 4-OHT concentrations, with the largest decrease at 1:3000. n = 3 independent isolations for all panels. Statistical significance was determined by repeated measures one-way ANOVAs (p values indicated on graphs) followed by Dunnett’s multiple comparisons test (α = 0.05). Values are mean ± standard deviation.
