Figure 1. Labeling and sorting Tsc1^fl/fl PCs show predominance of downregulation gene expression.

(a) Immunocytochemical characterization of wild-type control L7-GFP⁺ mouse PCs on P21 mouse cerebellum. Cell nuclei were stained with Hoechst. GFP⁺ PCs had dim expression of phosphorylated S6 (red). Scale bar 50 μm. (b) Immunocytochemical characterization of Tsc1^fl/fl L7Cre⁺L7-GFP⁺ mouse PCs on P21 mouse cerebellum. Cell nuclei were stained with Hoechst. GFP⁺ PCs were strongly positive for pS6 (red). Scale bar 50 μm. (c) Heatmap of differentially expressed genes in mutant (Tsc1^fl/fl; N = 4) vs control (Tsc1^+/+; N = 4) PCs (FC > 2 and p-value<0.01; n = 679 genes). Each row of the heatmap represents the scaled expression of one gene, where red corresponds to higher relative expression and blue corresponds to lower relative expression. (d) This volcano plot shows the relationship between fold change, calculated Mutant-Control, and p-value across all genes. A majority of genes in this dataset demonstrate downregulation in the mutant PCs compared to control.

Figure 1—figure supplement 1. Gating strategy for FACS isolation of GFP labeled PCs.

The gating for isolation of GFP-positive PCs is shown for (a) Tsc1^+/+ L7GFP and (b) Tsc1^fl/fl L7Cre;L7GFP animals. Cells were identified from debris based on the forward and side scatter (P1). Single cells were identified using FSC-H (P2) and SSC-H/SSC-W (P3). Finally, GFP-positive cells were selected and sorted into separate tubes (gate: POS).