Fig. 3

Mutant PKCγ forms cytoplasmic aggregates in iPSCs. a PRKCG mRNA expression in control and patient iPSC lines. RNA extracted from fetal and adult human cerebellum was included as positive controls. PRKCG is not expressed in peripheral blood mononuclear cells (PBMCs) according to data from GTEx, BioGPS, and CGAP SAGE, and thus, RNA extracted from PBMCs was used as negative control. PRKCG gene expression levels were normalized to housekeeping gene β-actin, and are shown relative to negative control. b PKCγ protein expression in control and patient iPSC lines. Actin: loading control. c Immunostaining of iPSC lines for PKCγ. Specificity of the anti-PKCγ antibody was confirmed by peptide absorption assay (top left panel). Small punctate staining of PKCγ (white solid arrowheads) was observed in the cytoplasm of control iPSCs and SCA14 iPSCs, while large cytoplasmic aggregates (white arrows) were only present in SCA14 iPSCs. Cell nuclei are visualized by Hoechst staining. Scale bar: 10 μm. d PKCγ formed significantly larger aggregates in SCA14 iPSCs compared to control iPSCs (n = 3, ****p < 0.0001, ANOVA followed by Bonferroni’s post-hoc test). e Treatment with 400 nM PMA, a potent PKCγ activator, both wildtype and mutant PKCγ aggregates increased in size. Compared to control, PKCγ formed significantly larger aggregates in SCA14 iPSCs following PMA treatment (n = 3, ****p < 0.0001, two-way ANOVA followed by Bonferroni’s post-hoc test)