Subunit c accumulation in homozygous Cb Cln3Δex7/8 cerebellar precursor cells a. Subunit c immunostaining and autofluorescence of 7-day confluency-aged wild-type and homozygous CbCln3Δex7/8 cells is shown. Wild-type cultures (CbCln3+/+) exhibited limited subunit c immunostaining and autofluorescence. However, CbCln3Δex7/8/Δex7/8 cells contained numerous subunit c puncta. Autofluorescence (7 days AF) was also significantly elevated (right panels), although limited overlap with subunit c puncta was observed (arrows). 40 × magnification. b. Immunoblot analysis of subunit c protein at sub-confluency or 7-day confluency incubation is shown. Total protein extracts from sub-confluency wild-type (+/+) and homozygous mutant (Δex7/8/Δex7/8) cultures contained approximately equal levels of subunit c protein (α-sub c). 7-day confluency extract from homozygous CbCln3Δex7/8 cells (Δex7/8/Δex7/8) had elevated levels of subunit c protein (~1.5X), relative to wild-type extract (+/+). Protein levels were normalized to cytochrome c oxidase subunit IV (α-cox4). c. TEM analysis of inclusions in 7-day confluency-aged homozygous CbCln3Δex7/8 cells is shown. A large autophagosome contained by double membrane (arrows) is filled with degenerating mitochondria (Md), electron dense cores (left and right of *) and other smaller vesicular structures. A large electron-dense inclusion, with a lipofuscin (Ln) appearance, is also present. M, mitochondria. 10,000 × magnification.