Supplementary Material for: CCP1 loss in bone marrow mesenchymal stem cells inhibited osteogenic differentiation by enhancing microtubule glutamylation

Cytosolic carboxypeptidase 1 (CCP1) is a deglutamylase that antagonizes polyglutamylation. Mutations in human CCP1 gene cause a severe disease known as childhood-onset neurodegeneration with cerebellar atrophy (CONDCA), which is characterized by marked growth retardation. However, the role and mechanisms of CCP1 in skeletal development remain unclear. In this study, we used CCP1 knockout (CCP1-KO) mice to assess bone mass changes by micro-CT, HE, alkaline phosphatase (ALP) staining, tartrateresistant acid phosphatase (TRAP) staining and immunofluorescence staining. Changes in osteogenic differentiation, proliferation and migration capacity of bone marrow mesenchymal stem cells (BMSCs) were assessed by ALP, alizarin red (ARS) staining, quantitative real-time PCR (qRT-PCR), EdU staining and cell scratching assay. Then, tubulin glutamylation and primary cilia of BMSCs after deletion of CCP1 was analyzed by western blot (WB) and immunofluorescence staining. Finally, CB839, an inhibitor of glutamine metabolism, was used to detect changes in the osteogenic differentiation ability and primary cilia of BMSCs after reducing the elevated glutamylation level. CCP1-KO mice exhibited phenotypes relevant to humans, including reduced body size, decreased bone mass, and reduced bone density during growth and development. CCP1 deficiency impairs the proliferation, migration and osteogenic differentiation of BMSCs. Meanwhile, the number of pre-osteoblasts derived from BMSCs is decreased, leading to impaired osteogenesis. At the cellular level, CCP1 loss results in aberrant tubulin glutamylation, increased microtubule glutamylation, and shortened primary cilia in BMSCs. Finally, reduction of abnormally elevated tubulin glutamylation was efficacious for promoting osteogenic differentiation of BMSCs and restoring primary cilia length of BMSCs. We propose that CCP1 plays a critical role in regulating BMSCs differentiation and promotes osteogenesis by modulating the post-translational modifications (PTM) of tubulin, with a view to provide new targets for the prevention and treatment of hard tissue diseases.