Fig. 2: GAG chain elongation is impaired in TMEM165-deficient cells. | Cell Death & Disease

Fig. 2: GAG chain elongation is impaired in TMEM165-deficient cells.

From: TMEM165 a new player in proteoglycan synthesis: loss of TMEM165 impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans and triggers early chondrocyte differentiation and hypertrophy

Fig. 2

A PG anabolism evaluation in wild-type and tmem165-knockout mouse ATDC5 cells by measurement of the incorporation rate of [35S]-sulfate into the GAG chains (n = 3). B SDS-PAGE and autoradiography analysis of neosynthesized radiolabelled PG-GAG chains in wild-type and tmem165-knockout mouse ATDC5 cells (n = 3). C Detection of decorin in conditioned medium of wild-type and tmem165-knockout mouse ATDC5 cells and (D) in wild-type and TMEM165-knockout HEK293 cells (n = 3). E Detection of decorin S34A mutant lacking GAG chain in conditioned medium of wild-type and mutant mouse ATDC5 cells (n = 3). F Analysis of the sensitivity to degradation by chondroitinase ABC of GAG chains of decorin in conditioned medium of wild-type and tmem165-knockout mouse ATDC5 cells (n = 3). G SDS-PAGE and autoradiography analysis of neosynthesized radiolabelled GAG chains primed with 4MU-Xyl in wild-type and tmem165-knockout mouse ATDC5 cells (n = 3). H Detection of HA-syndecan 4 in cell lysates of wild-type and tmem165-knockout mouse ATDC5 cells transfected with HA-syndecan-4 expression vector. β-actin was used as loading control. (n = 3) I Immunofluorescence analysis of cell surface HS GAG chains using anti-HS specific antibodies (green) and of the expression of TMEM165 (red) in wild-type and tmem165-knockout mouse ATDC5 cells. The nucleus was stained with DAPI (blue). Digital images were captured with an inverted microscope, Leica DM13000. Representative images from three independent experiments are shown. Scale bar: 50 μm.

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