HAP1 (Wild-type and SLC10A7 KO cells) were obtained from Horizon Discovery (HZGH005271c011). Patient fibroblasts from P1 and P2 were obtained from Pr Dirk Lefeber, Radboud University, The Netherlands, and patient fibroblasts P3, P4, and P5 from Pr Valérie Cormier-Daire, Necker Hospital, Paris, France. HAP1 cells were harvested in IMDM and fibroblasts in DMEM (Lonza, Basel, Switzerland), supplemented with 10% Fetal Bovine Serum (FBS) (Pan, Pan Biotech), in a humidified atmosphere with 5% CO2 at 37 °C. For drug treatment, cells were incubated with MnCl2 or Ac3Bn-α-GalNAc (Sigma-Aldrich, St Louis, Missouri, USA) for different times/ concentrations as indicated in the figures and their legends.

For cell lysate, cells were scrapped on ice, in PBS (Euromedex, Souffelweyersheim, France), placed in Eppendorf tubes and centrifugated for 10 min at 4 °C. The supernatants were eliminated, and cell pellets were lysed by 20 aspirations/discharges with RIPA buffer [Tris/HCl 50 mM pH 7.9, NaCl 120 mM, NP40 0.5%, EDTA 1 mM, Na3VO4 1 mM, NaF 5 mM] supplemented with a protease cocktail inhibitor (Roche Diagnostics, Penzberg, Germany), and vortexed twice for 10 s. Samples were then centrifugated for 30 min at 4 °C, 20,000 g. Protein concentration contained in the supernatant was estimated with the micro-BCA Protein Assay Kit (Thermo Scientific). For proteins coming from cell culture media, proteins were precipitated by adding a methanol volume corresponding at 3 times the cell culture media volume, and 0.75 volume of chloroform. After vortexing for 15 s, 2 volumes of water were added and tubes were centrifugated for 15 min at 4 °C, 869 g. The supernatant above proteins was discarded, and the sample was mixed with 2.25 volumes of methanol and centrifugated in the same conditions. The whole supernatant was discarded, and the protein pellet was resuspended in RIPA, and transferred to a microtube, before 30 min of centrifugation at 20,000 g, 4 °C. Fifteen µL of the supernatant were mixed with loading NUPAGE blue on a gel. Western blot samples of cells were prepared by mixing 10 (LAMP2, TGN46) or 20 µg of protein lysate (C1GALT1, COSMC) with water to reach 15 µL, and 5 µL of NuPAGE LDS (Invitrogen Carlsbad, California) supplemented with 16% of beta-mercaptoethanol were added. Samples were heated for 10 min at 95 °C (except for COSMC), separated on 4–12% BisTris gels (Invitrogen, Carlsbad, California), and then transferred with the iBlot 2 system (Program 0–7 min, Invitrogen, Carlsbad, California). Membranes were blocked for 1–3 h in TBS-Tween-0.05% + 5% dry milk or BSA (fraction V, Roche Diagnostics, Penzberg, Germany). Primary antibodies (see antibodies table) were incubated overnight in the blocking solution, at 4 °C under agitation. The day after, membranes were washed three times with TBS-Tween, and secondary antibodies were diluted in the blocking solution and incubated for 1 h at room temperature. Membranes were washed five times for 5 min with TBS-Tween. Signal was detected with a chemiluminescence reagent (ECL 2 Western Blotting Substrate or SuperSignal West Pico PLUS chemiluminescent Substrate, Thermo Scientific) in a dark room on imaging film (GE Healthcare, Little Chalfont, United Kingdom).

Cells harvested on glass coverslips were fixed with 4% paraformaldehyde for 20 min, washed 3 times and coverslips were mounted in a wet chamber. Cells were permeabilized with PBS + 0.5% Triton-X100 for 10 min and washed with PBS three times, then blocked for 1 h in blocking buffer [0.2% gelatin, 2% Bovine Serum Albumin (BSA), 2% Fetal Bovine Serum (FBS) (Lonza) in PBS]. Primary antibodies were diluted at 1/100 in blocking buffer and incubated for 1 h. After 3 washes with PBS, secondary antibodies (GAR-Alexa Fluor 568 or GAM-Alexa fluor 488) were incubated for 1 h at 1/600. Cells were washed 3 times with PBS, and nucleus were stained for 10 min with 5 µg/mL DAPI blue. Cells were then mounted with 6 µL of Mowiol on glass slides after 10 rinses with deionized water. Fluorescence was detected through an inverted Zeiss LSM780 or LSM700 confocal microscope. Acquisitions were done using the ZEN pro 2.1 software (Zeiss, Oberkochen, Germany), and images were treated with ImageJ (Fiji).

For fluorescent lectin: coverslips were fixed and mounted in a wet chamber as described in the previous section. Paraformaldehyde was neutralized with 50 µM NH4Cl for 15 min, and coverslips were washed three times. Permeabilization, if needed, was performed with a mix of PBS + 0.1% BSA + 0.075% saponin, incubated for 15 min. Lectin (Vicia Villosa Lectin (VVL) recognized the O-GalNAc (= Tn Antigen) coupled to Fluorescein-Vector Laboratories, Newark, California, United States) were then incubated at 1/1000 in PBS + 0.1% BSA for 1 h, protected from light. After 3 washes, DAPI, mounting, and image acquisition were performed as described in the “immunofluorescence” section. For lectin-blot, samples preparation and migration were conducted as described in the Western blot section. Proteins were transferred on nitrocellulose membrane with the liquid Invitrogen (Carlsbad, California) transfer system, for 1 h at 10 V. Membranes were blocked 1 h in filtered 2% PVP—0.05% TBS-Tween. Biotinylated lectins (Vector Laboratories, Newark, California, United States) were incubated at 2 µg/mL in the same buffer for 45 min, and then washed 3 times for 15 min with TBST. Antibiotin-HRP antibodies (Sigma Aldrich, St-Louis, Missouri, United States) were diluted in the blocking buffer at 1/10000, incubated for 1 h and washed 6 times for 15 min with TBST. Signal was detected with chemiluminescence reagent (ECL 2 Western Blotting Substrate or SuperSignal West Pico PLUS chemiluminescent substrate, Thermo Scientific) using a CCD camera (FUSION Solo, Vilber Lourmat, France) and the FUSION-Capt Solo software for acquisition.

Patient fibroblasts were grown on glass coverslips to carry out cell imaging experiments. Ratiometric dye Fura-2/AM (F1221, Invitrogen) was used as a Ca2+ indicator. Cells were loaded with 2 μM Fura-2/AM for 45 min at 37 °C and 5% CO2 in corresponding medium and subsequently washed three times with external solution containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 5 Glucose and 10 Hepes (pH 7.4). The coverslip was then transferred in a perfusion chamber on the stage of Nikon Eclipse Ti microscope. Fluorescence was alternatively excited with a monochromator (Polychrome IV, TILL Photonics Gmbh) at 340 and 380 nm (for Ca-imaging experiments). Then, fluorescence was captured at 510 nm by a QImaging CCD camera (QImaging). Acquisition and analysis were performed with MetaFluor 7.7.5.0 software (Molecular Devices Corp.).

The protocol used for Bn-GalNAc glycosylation status analysis by mass spectrometry (MALDI-QIT-TOF, Matrix-Assisted Laser Desorption/Ionisation-Quadrupole Ion Trap Time-Of-Flight) was described in Durin, Houdou, 2022 [14]. The same protocol was used, except for HAP1 cells that were cultured in IMDM complemented with 5% FBS, instead of DMEM.

The deglycosylation assay was performed with the Agilent Enzymatic Deglycosylation Kit for N-Linked and Simple O-Linked Glycans (GK80110) and Agilent Extender Kit for Complex O-Linked Glycans (GK80115), according to manufacturer’s instructions (Agilent, Santa Clara, CA 95051 United States). Briefly, cells were lysed in RIPA buffer as described above, without protease inhibitors, in order to reach a concentration of 100 µg of protein in a maximum volume of 30 µL. Fifty to 100 µg of protein were heated at 100 °C for 10 min in 10 µL of incubation buffer supplemented with 2.5 µL of denaturation buffer and cooled down on ice. 2.5 µL of detergent solution and 1 µL of required enzymes were added, and samples were incubated for at least 3 h at 37 °C under agitation. The adequate volume to reach 10 µg of proteins per sample was then submitted to the Western blot protocol detailed above.

Antibody (specie)

Gel and samples preparation

Buffer

Dilution primary

Dilution secondary

Western blot

TGN46-Biorad (Sheep) #AHP500G

Precast – 10 µg-Heated

Milk 5%

1/1000 Overnight

Donkey anti-Sheep (Daiko) 1/10000

LAMP2-Santa Cruz (Mouse) #sc-18822

Precast – 10 µg-Heated

Milk 5%

1/2000 Overnight

Goat anti-Mouse (Daiko) 1/20000

COSMC–Santa Cruz (Mouse) #sc-271829

Homecast (20 µg)-Heated

BSA 5%

1/500 Overnight

Goat anti-Mouse (Daiko) 1/20000

C1GALT1-Santa Cruz (Mouse) #sc-100745

Precast-20 µg-Heated

Milk 5%

1/1000 Overnight

Goat anti-Mouse (Daiko) 1/20000

β-Actin – Sigma Aldrich (Mouse) #A1978

Homecast or Precast-10 to 20 µg-Heated or not

Milk or BSA 5%

1/10000 Overnight or 1 h

Goat anti-Mouse (Daiko) 1/20000

Cab45-Sigma Prestige (Rabbit) #HAP011249

Homecast-10 µg-Not heated

BSA 5%

1/1000 Overnight

Goat anti-Rabbit (Daiko) 1/5000

Antibody (specie)

Dilution primary

Dilution secondary (Alexa Fluor)

Immunofluorescence

GM130 – BD Bioscience (mouse) #610,822

1/100

GAM488 1/600

Giantin – Biolegend Covance (rabbit) #909,701

1/100

GAR568 1/600

Cab45 – Sigma Prestige (Rabbit) #HAP011249

1/100

GAR568 1/600