Fig 2: Bioinformatics prediction for c.1284+2del and TA clone-sequencing results of EXT1 mRNA. (A) Predictive output of c.1284+2del in EXT1 from CRYP-SKIP, the input EXT1 sequences involved exon 4 (capital letters) and adjacent intronic sequences (lower case letters). (B) TA cloning and sequencing results of the EXT1 gene. The arrows indicate the borders of adjacent exons. EXT1, exostosin glycosyltransferase 1; WT, wild type; MT, mutated type.

Fig 3: EXT1 localizes in ER tubules and sheet matrices.(A and B) STED (A) and SIM (B) images of Cos7 cells expressing SYFP2-EXT1 and mEmerald-EXT1, respectively. Boxed regions illustrate the tubular (subpanel a) and the cisternal (subpanel b) ER. Scale bars, 4 µm. (C) Confocal fluorescence microscopy of Cos7 cells transiently expressing SYFP2-EXT1 (green) and endogenous ER marker PDIA3 (red). The subpanels show the individual and merged channels and the colocalized pixel map (CPM). Scale bar, 4 µm. (D to F) As in (C) but coexpression of SYFP2-EXT1 (green) and indicated ER markers (red). Scale bars, 4 µm. (G) Live imaging of shCTRL and shEXT1 Cos7 cells stably expressing indicated ER markers. Scale bars, 4 µm. Boxed region illustrates the ER. (H) HS endogenous staining (red) of Cos7 cells. Scale bars, 5 µm. (I) Endogenous staining (red) of Cos7 cells with Calnexin antibody. Boxed regions magnified illustrate a zoom of circular, vesicle-like structures that appear following EXT1 k.d. Scale bars, 4 µm. See also fig. S6.

Fig 4: 2D plot profiling for quantification and correlation of spatial gradients of IF signals from primary antibodies against HS GAG, HS GAG biosynthesis enzymes and common leukocyte antigen (CD45) in histological sections of gingiva from patient with advanced generalized periodontitis (sample: IP-DJD-2CHP). (Magnification: × 10; scale bar: 1000 µm). Spatial gradients of IF signals are plotted simultaneously on a single T-D plot (A) to devise the regression model. The purpose of the model is to disclose to how the presence of inflammatory cell infiltrate correlates with the expression of HS GAG and HS GAG biosynthesis enzymes. The spatial gradient for CD45 IF signal is set as dependent variable (y), whereas the spatial gradients for IF signals from HS 3G10, HS 10E4, EXT1, EXT2, NDST1 and NDST1 are designated as independent variables (x). Based on the regression function, a predicted CD45 spatial gradient is calculated (MODEL CD45) and plotted with the actual values of CD45 spatial gradient on several different plots (B–D) to check for the goodness of fit. The model is statistically significant (a = 1 × 10–8, P < 1 × 10–8) and reveals that the presence of inflammatory infiltrate can be well correlated with the expression of HS GAG and HS GAG biosynthesis enzymes (linear model: R = 0.95576; R2 = 0.91348; polynomial curve fitting: R = 0.96883; R2 = 0.93844). As visualized on the bar-chart of correlation coefficients (E), the deviation of the model (C,D) can be attributed to the low individual correlation of particular independent variables such as HS 3G10 (R = 0.32808; R2 = 0.10763), HS 10E4 (R = 0.02445; R2 = 0.00059), EXT2 (R = 0.02191; R2 = 0.00048) and NDST1 (R = 0.07752; R2 = 0.00601). IF signals of HS 3G10, HS 10E4, EXT2 and NSDT1 have larger expression domains and display different patterns than those of EXT1 and NDST2 (more confined to stromal compartment with CD45) (top row). Thus, the spatial gradients of EXT1, NDST2 display stronger correlation (EXT1–R = 0.52064; R2 = 0.27107) (NDST2–R = 0.55022; R2 = 0.30275) with CD45. (Image created in Adobe Photoshop CC 2014, ver. 6.3; https://www.adobe.com/products/photoshop.html).

Fig 5: Intensity distribution hotmaps for syndecans and HS biosynthesis enzymes in human incisor tooth germ during the late bud (7/8 w) (A,E,I,M,Q,U), cap (11/12 w) (B,F,J,N,R,V), and early bell stage (14 w) cervical loop (C,G,K,O,S,W) and inner enamel epithelium area (D,H,L,P,T,X). Far Left: colorized pixel intensity scale paired with seven corresponding mean values from Kodak No. 3 step calibration tablet. Arrows point to magnified framed areas on hotmaps for Sdc1 (A), Sdc2 (F), Sdc4 (J), EXT1 (P), NDST1 (S), and NDST2 (W) at different stages of tooth development. Magnification: (A–X) × 40, scale bar: 60 µm. Intensity range (pixel value): 15–30 (dark blue), 30–45 (bright blue), 45–55 (dark green), 55–70 (bright green), 70–85 (dark red), 85–95 (bright red), and 95–255 (yellow).