DRG Pituitary Cerebral Cortex

Transcription

1 Liver Spinal cord Pons Atg5 -/- Atg5 +/+ DRG Pituitary Cerebral Cortex WT KO Supplementary Figure S1 Ubiquitin-positive IBs accumulate in Atg5 -/- tissues. Atg5 -/- neonatal tissues were fixed and decalcified. Whole-mount paraffin sections were prepared and stained with an anti-ubiquitin antibody (1B3). Representative regions of liver, anterior horn of the spinal cord, pons, dorsal root ganglia, anterior lobe of pituitary gland, and cerebral cortex are shown. Scale bar, 10 μm.

2 a 1 kb Wild-type allele X X K exon 2 exon 3 ATG X S H probe 9kb exon 4 Targeting vector lox Neo lox DT-A pbsk Flox allele X X lox K Neo S probe 11kb Deleted allele lox 15kb probe b exon 3 Wild-type allele A B Flox allele D Neo C B E F Cre recombinase Deleted allele D B Supplementary Figure S2 Targeting and genotyping strategies for the Atg5 flox/flox mice. (a) The restriction map of the wild-type Atg5 allele, the targeting construct, flox allele, and the deleted allele after Cre-mediated recombination. Black boxes indicate exons 2-4. Probes used for Southern blot analysis are indicated by open boxes. The Eco and KpnI-digested fragments detected by probes are depicted. Restriction enzymes are as follows: X; XbaI, ; Eco, H; HindIII, S; SpeI, K; KpnI. (b) The positions of PCR primers in the wild-type Atg5 allele, the targeting construct, the flox allele, and the deleted allele. Primers A and B were used for amplification of the wild-type Atg5 allele; primers C and B for the flox allele; primers D and B for the deleted allele; and primers E and F for detection of the Cre recombinase gene.

3 a Brain Pancrease +/+ Deleted allele Flox allele Wild allele b E9.5 E15.5 P0 Flox Wild Deleted Cre c E15.5 P0 Atg12-5 LC3-I LC3-II β-actin Supplementary Figure S3 Atg5 gene deletion in Atg5 flox/flox ;Nestin-Cre mice. (a, b) Genomic DNA extracted from Atg5 +/+ and Atg5 flox/flox ;Nestin-Cre mice at eight weeks of age (a), and embryos (b) were analyzed by Southern blot analysis (a) and PCR (b). The probe and primers used were indicated in Supplementary Figure S2. (c) Immunoblot analysis of Atg5 and LC3 performed as described in Figure 1.

4 Pons DRG Cerebral cortex Ctrl Supplementary Figure S4 Ubiquitin-positive IBs accumulated in Atg5 flox/flox ;Nestin-Cre neonates. Tissue sections of pons, DRG, and cerebral cortex were prepared from day 0 neonates of Atg5 flox/flox ;Nestin-Cre and control (ctrl; Atg5 flox/+ ;Nestin-Cre) mice as described in Supplementary Figure S1 and stained with anti-ubiqutin antibody (1B3). IBs accumulated in the pons and DRG, but not in the cerebral cortex. Scale bar, 10 μm.

5 Supplementary Figure S5 Larger images of the electron microscopic data shown in Fig. 3c.

6 Heart f/f;cag Brain Liver Skeletal Muscle Deleted allele Flox allele Supplementary Figure S6 Southern blot analysis of genomic DNA extracted from various tissues of Atg5 flox/flox ;CAG-Cre mice.

7 Ctrl shorter exposure female Ctrl male Ctrl female longer exposure Ctrl male Polyubiquitin GSK-3β Supplementary Figure S7 Immunoblot analysis of brain lysates. Whole-brain lysates from control (ctrl; Atg5 flox /+ ;Nestin-Cre) and Atg5 flox/flox ;Nestin-Cre mice at six weeks of age were subjected to immunoblot analysis with antibodies to polyubiquitin (FK2) and GSK-3β (loading control). The right panel of the polyubiquitin blot is a longer exposure of the left panel. Accumulation of ubiquitinated proteins was very similar in male and female mice.

8 Autophagy normal normal proteins misfolded proteins ubiquitinated proteins Proteasome soluble Autophagy Autophagy deficient normal proteins misfolded proteins ubiquitinated proteins soluble Autophagy inclusions Supplementary Figure S8 Model for the role of autophagy in protein quality control. Under normal conditions, autophagy constitutively degrades diffuse cytosolic proteins in basically a non-selective manner, whereas ubiquitinated proteins are efficiently degraded by the proteasome. If autophagy is blocked, general protein turnover is impaired, resulting in the accumulation of misfolded and ubiquitinated proteins, which ultimately aggregate within the cytoplasm.