Fig. 7: BMP signaling is activated in TMEM165-deficient cells.

A Detection of phosphorylated Smad1, 5, 9 (pSmad1,5,9) and total Smad in cell lysates from wild-type and tmem165-mutant mouse ATDC5 cells and (B) from normal fibroblasts and TMEM165-deficient CDG patient fibroblast cells (n = 3). C Fold changes of Id1 expression in tmem165-knockout cells normalized to wild-type mouse ATDC5 cells. D Fold changes of BMP reporter activity in tmem165-knockout cells normalized to wild-type mouse ATDC5 cells. E Fold changes of BMPR1A, BMPR1B and BMPR2 expression in tmem165-knockout cells normalized to wild-type mouse ATDC5 cells. F Detection of BMPR2 in cell lysates of wild-type and tmem165-knockout mouse ATDC5 cells. β-actin was used as loading control (n = 3). G Fold changes of BMPR1A, BMPR1B and BMPR2 expression in TMEM165-deficient fibroblasts normalized to normal fibroblast cells. H Detection of BMPR2 in cell lysates of normal fibroblasts and TMEM165-deficient CDG patient fibroblast cells. β-actin was used as loading control (n = 3). I Fold changes of Noggin expression in tmem165-knockout mouse ATDC5 cells normalized to wild-type ATDC5 cells and (J) in TMEM165-deficient CDG patient fibroblasts normalized to normal fibroblast cells. qPCR values were normalized for the housekeeping gene ribosomal protein S29 and are expressed as the relative expression compared with control. Data are expressed as mean ± S.D. Statistical analysis was performed with an unpaired Student’s t test (n = 3; *p < 0.05; **p < 0.01). K Detection of phosphorylated Smad2 (pSmad2) and Smad1, 5, 9 (pSmad1,5,9) and of total Smad in cell lysates from wild-type and tmem165-mutant mouse ATDC5 cells cultured in medium with or without Mn²⁺ supplementation. β-actin was used as loading control (n = 3). Representative images from three independent experiments are shown.