Fig 2: DEPTOR controls TAZ subcellular localization. (A–B) Co-IP of DEPTOR and TAZ. (A) N-terminal HA tagged TAZ and N-terminal Flag tagged DEPTOR were cotransfected into HEK293T cells. After transfection cells were starved O/N and then treated with 10% FBS and MG132 for 4 hours. TAZ was pulled down using anti-HA magnetic beads and the interaction with DEPTOR was detected by western blot using an anti-Flag antibody. (B) N-terminal HA tagged TAZ and N-terminal Flag tagged 13xS/T-A DEPTOR were cotransfected into HEK293T cells. After transfection cells were starved O/N. IP was performed as in (A). (C) Western blots of Control and shDEPTOR1/2 lines treated with Dox. Cells were starved O/N and then treated with 10% FBS for two and 4 hours. Numbers indicate quantification of bands using Fiji. (D) Immunofluorescence of TAZ in Dox treated Control and shDEPTOR1/2 lines under starvation and after 4 h 10% FBS treatment. (E) Luciferase reporter assay of YAP/TAZ reporter (8xGTIIC) in Control and shDEPTOR1/2 lines under starvation. Graphs represent means ± SEM. N = 30. Student’s t-test, ****p < .0001. (F–G) Gene expression analysis of YAP/TAZ target genes CTGF (F) and CYR61 (G) under starvation, with and without dox as measured by qPCR. Graphs represent means ± SEM. N = 4. Student’s t-test, *p < .05, ****p < .0001. Dox, doxycycline. Scale bars: 15 µm.

Fig 3: DEPTOR-DVL2 interaction regulates TAZ shuttling between nucleus and cytoplasm. (A) Co-IP of DEPTOR and DVL2. N-terminal HA tagged DEPTOR and N-terminal Flag tagged DVL2 were cotransfected into HEK293T cells. After transfection cells were starved O/N and then treated with 10% FBS alone or in combination with MG132 for 4 h. DEPTOR was pulled down using anti-HA magnetic beads and the interaction with DVL2 was detected by western blot using an anti-Flag antibody. (B) Western blots of Control and shDEPTOR1/2 lines. Cells were starved O/N and then treated with 10% FBS for 4 h. (C) Western blot of DVL2 in Control and JMC fibroblasts. Cells were starved O/N and then treated with 10% FBS for two and 4 hours. (D) Co-IP of DEPTOR and PTH1R. N-terminal HA tagged PTH1R and N-terminal Flag tagged DEPTOR were cotransfected into HEK293T cells. After transfection cells were starved O/N and then treated with 10% FBS alone or in combination with MG132 for 4 h. DEPTOR was pulled down using anti-Flag magnetic beads and the interaction with PTH1R was detected by western blot using an anti-HA antibody. (E–F) Co-IP of PTH1R or TAZ and DEPTOR DEP and PDZ domains. (E) N-terminal HA tagged PTH1R and N-terminal Flag tagged DEP or PDZ domains were cotransfected into HEK293T cells. After transfection cells were starved O/N and then treated with 10% FBS for 4 h. DEP and PDZ domains were pulled down using anti-Flag magnetic beads and the interaction with PTH1R was detected by western blot using an anti-HA antibody. (F) N-terminal HA tagged TAZ and N-terminal Flag tagged DEP or PDZ domains were cotransfected into HEK293T cells. After transfection cells were starved O/N. DEP and PDZ domains were pulled down using anti-Flag magnetic beads and the interaction with TAZ was detected by western blot using an anti-HA antibody.

Fig 4: JMC and BOCD patients show aberrant differentiation of skeletal progenitors. (A–F) Masson Goldners staining of Control (A, D), JMC (B, E) and BOCD (C, F). Note the accumulation of fat and the small region of cartilage in the trabecular bone of JMC patients (magnification in B), and the invasion of chondrocytes in the trabecular bone of BOCD patients (magnification in C). Arrows point to calcified islands, asterisks indicate undermineralized trabecular bone. (G–I) Immunolocalization of ALP of Control (G), JMC (H) and BOCD (I). (J–L) Immunolocalization of AGGRECAN in Control (J), JMC (K) and BOCD (L). ALP staining demonstrates decreased bone mass in JMC and BOCD patients; AGGRECAN immunolocalization confirms the presence of cartilage in JMC and BOCD trabecular bone. (M–Q) Immunolocalization of DEPTOR in Control (M), JMC (N, P) and BOCD (O, Q). JMC, Jansen Methaphyseal Chondrodysplasia; BOCD, Blomstrand Chondrodysplasia; ALP, Alkaline phosphatase. Scale bars, 100 µm.