(A) GRASP KO increases the expression of HS synthesis enzymes while decreases CS synthesis enzymes. Results are based on RNA-Seq analysis of each cell line for indicated genes. (B) GRASP KO increases the protein level of HS synthesis enzymes while decreases that of CS synthesis enzymes. Cell lysate of indicated cells were analyzed for three HS synthesis enzymes EXTL3, EXT1 and EXT2, and a key CS synthesis enzyme GalNAcT1. Note the increased level of EXTL3 and decreased GalNAcT1 level in GRASP KO cells. Results are representative of three independent experiments. (C) Quantification of B. (D) GRASP KO decreases the protein level of the HS sulfation enzyme PAPSS2. Shown are representative Western blots of indicated proteins in the four cell lines from three independent experiments. (E) Quantitation of D. (F) Re-expression of GRASP proteins in GRASP KO cells corrects the expression level of HS and CS synthesis enzymes. Indicated cell lines were transfected with GRASP constructs and probed for EXTL3, GalNAcT1, GRASP65, GRASP55, GFP, and actin. The major enzymes EXTL3 and GalNAcT1 in 55KO and 65KO cells were rescued by expressing GRASP55-GFP or GRASP65-GFP, respectively, but not by GFP alone (lanes 4 & 7 vs. 3 & 6). (G) Re-expression of GRASP proteins corrects the HS and CS defects in GRASP KO cells. Confocal images of WT HeLa cells and GRASP KO cell lines transfected with indicated constructs followed by HS staining. The level of HS in 55KO and 65KO cells was decreased by the expression of GRASP55-GFP or GRASP65-GFP, respectively, but not by GFP alone. Note the different HS signals in cells expressing GRASP55- or GRASP65-GFP (asterisks) vs. non-transfected cells (arrows). (H-I) Quantification of G. Results are presented as mean ± SEM, statistical analysis was assessed by comparing KO cells to WT cells by student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001.