Scintillation fluid

Metabolic labeling of GAG chains of PGs was carried out using [³⁵S]-sulfate incorporation method as described by [27 (link)]. Briefly, subconfluent mouse ATDC5 cells grown in six-well culture plate were radiolabeled with 10 µCi/ml of [³⁵S]-sulfate (Perkin Elmer, Courtabœuf, France) overnight. Then, conditioned culture medium was collected, digested with papain (1 mg/ml), and [³⁵S]-labeled GAGs were precipitated by cetylpyridinium chloride (CPC) as described by [28 (link)]. When GAG chains were primed by 4MU-Xyl, cells were cultured in the presence of 10 µCi/ml of [³⁵S]-sulfate and 100 μM of 4MU-Xyl overnight, and radiolabeled GAG chains were directly precipitated from conditioned medium by CPC. The CPC precipitated radiolabeled GAGs were separated by SDS-PAGE on a 4–20% Tris/Glycine gel. The gel was dried and exposed to autoradiography film.
To measure the rate of sulfate incorporation into GAG chains of PGs, mouse ATDC5 cells were radiolabeled with 10 µCi/ml of [³⁵S]-sulfate for 6 h then, conditioned culture medium was collected and digested with papain (1 mg/ml). [³⁵S]-labeled GAG chains were precipitated by CPC dissolved in solvable and mixed in scintillation fluid (Perkin Elmer, MA, USA). The radioactivity associated with GAGs was measured by liquid scintillation counting (Packard, Rungis, France).

Human FGF9 was purchased from R&D Systems (Minneapolis, MN, USA). Scintillation fluid was purchased from PerkinElmer (Boston, MA, USA). Fetal bovine serum and M199 powder were purchased from Gibco (Grand Island, NY, USA). Nitroblue tetrazolium, β-nicotinamide adenine dinucleotide, dihydroepiandrosterone, EDTA, ethylene glycol tetraacetic acid, sodium pyrophosphate, β-glycerophosphate and sodium orthovanadate were purchased from Sigma (Seelze, Germany). Waymouth's MB752/1, PD98059, SP600125, SB203580, and wortmannin were purchased from Sigma (St. Louis, MO, USA). 1,2,6,7-3H(N)-testosterone (70.00 Ci/mmol) and 1,2,6,7-3H(N)-progesterone (70.00 Ci/mmol) in 0.25 ml ethanol were purchased from DuPont-New England Nuclear (Boston, MA, USA). Antibodies against phospho-ERK1/2, phospho-p38, phospho-JNK, and phospho-Akt were purchased from Cell Signaling (Beverly, MA, USA). Antibody against β-actin was purchased from Sigma (St. Louis, MO, USA).

Synechocystis strains (wild-type and strains harboring the xylAB genes) were grown as described above, until the OD₇₃₀ reached ∼0.8. Then, 10 ml of the cells were washed thrice with BG-11 medium and OD₇₃₀ was adjusted to 0.6. Next, 1 ml of the cultures was grown in the presence of 400 nM ¹⁴C-labelled xylose [D-(1-¹⁴C)] (American Radiolabeled Chemicals) in 10 ml snap-capped glass tubes under similar growth conditions for 1 h. Nine milliliters of BG-11 medium was added to the cultures and they were filtered through a 0.45 μm pore-size nitrocellulose membrane (Bio-Rad). The membranes were washed thrice with 10 ml BG-11 medium, air dried, and suspended in 10 ml scintillation fluid (PerkinElmer) for 24 h. Radioactivity was measured using an LS 6500 scintillation counter (Beckman).

Proteoglycan synthesis was measured by ³⁵S-sulfate incorporation as described by De Vries et al., [17 (link)]. Briefly, fibroblasts were grown in 6-well culture plate until 80% confluence then treated with either 4-MU4-deoxy-xyloside or 4-MU-xyloside in the absence or presence of TGF-β1 (5 ng/ml) for 24 h. At 6 h before the end of the treatment, cells were radiolabeled with 10 μCi/ml of ³⁵S-sulfate (Perkin Elmer, Courtabœuf, France). Culture medium was collected, digested with papain (1 mg/ml) and ³⁵S-labeled GAGs were precipitated by cetylpyridinium chloride (CPC), dissolved in solvable and mixed in scintillation fluid (Perkin Elmer, MA, USA). The radioactivity associated with GAGs was measured by liquid scintillation counting (Packard, Rungis, France).
To analyze endogenous PG-GAG chains or GAG chains primed with 4-MU-xyloside, cells were metabolically labelled with 10 μCi/ml of ³⁵S-sulfate (Perkin Elmer, Courtabœuf, France) for 24 h in the presence or not of 4-MU4-deoxy-xyloside. Culture medium was collected, digested with papain (1 mg/ml) and ³⁵S-labeled GAGs were precipitated by CPC. Isolated GAGs were analyzed by SDS-PAGE in 4–12% Nu-PAGE gel. After migration the gel was dried and exposed to autoradiography film. The radioactivity corresponding to GAGs was normalized with the amount of DNA of corresponding cells.

The effect of LNCs on cell proliferation was assessed by [³H]thymidine assay. Briefly, cells were plated at a density of 5 × 10⁴ cells/well (using 24 well plates) and incubated for 24 h. LNCs of different concentrations were added and the incubations prolonged for 24 h. After aspirating the media, [³H]thymidine (3 μg/ml in serum free media; 75 μCi/ml) was added and incubated for 30 min at 37 °C. The solution was then replaced with 5% TCA (800 µl/well) followed by incubation for 10 min at room temperature to precipitate nucleic acids and proteins. After washing once more with TCA, the TCA solution was aspirated and 0.1 M KOH (200 µl) was added to the wells followed by incubation for 15 min at room temperature to solubilize the precipitated nucleic acids and proteins. The solution was transferred to scintillation vials, mixed with 3 ml of scintillation fluid (Perkin Elmer, USA) and the radioactivity was measured for 60 s using a scintillation counter (Tri-Carb 2100TR, Packard Bioscience, USA).

Activity of purified FMDV 3D^pol was assessed by the ability of the protein to incorporate [α-³²P] UTP into nascent RNA transcripts, as previously described^{29 (link),44 (link)}. The final concentration of 3D^pol that was included in the assays was 18.52 µM. Reactions were stopped by the addition of 1.5 µl 500 mM EDTA at the indicated times. For scintillation, 2 µl of each reaction was blotted onto filter paper (Whatmann) and allowed to dry prior to being washed as described previously^{44 (link)}. The dry filter paper was exposed to film for imaging before the individual dots on the filter paper were excised and scintillated with the addition of 3 ml scintillation fluid (Perkin Elmer). Radioactive counts were measured as scintillation counts per minute.
For glutaraldehyde crosslinking, 3D^pol activity assays, substituting [α-³²P] UTP with 4 mM UTP were employed. Samples were incubated with 5 mM glutaraldehyde at 30 °C for increasing time periods and reactions stopped by the addition of 30 µL 2x Laemmli buffer. The samples were denatured and analysed by gradient SDS-PAGE (12–4% resolving gels, and 4% stacking gel). Immunoblotting was performed as described previously^{45 (link)} with primary rabbit anti-3D 397 polyclonal antibody (kind gift from Francisco Sobrino, Centro De Biologia Molecular Severo Ochoa, Madrid, Spain) and detected with anti-rabbit HRP (Sigma-Aldrich).