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, 4 and lysed by two pulse-mode sonication burst cycles of 30 seconds each. Samples were centrifuged for 30 minutes at 4°C, 16 000g, Monolayer cell cultures were resuspended in 1 mL of 10 mM Tris, 1 mM EDTA, 0.1 M NaCl, vol.33258, p.3569
, Fluorometric measurements (348 nm excitation wavelength, 456 nm absorption wavelength) were performed with the
Thermo Scientific) running Skan it 2.4.3 software. Absolute quantification was determined using standard curves prepared from calf thymus DNA (D3664-2MG Sigma) ,
, RNA extraction and reverse transcription-polymerase chain reaction analysis. Total RNA was isolated from ATDC5 cells using RNeasy mini kit® (74104 Qiagen). 100ng of total RNA were reverse-transcribed using M-MLV reverse transcriptase (28025021 Invitrogen) with random hexamer primers
, Real-time quantitative polymerase chain reaction
, Real time quantitative PCR was performed with standard curve calibration, using the following primers : RPS29_For, GGAGTCACCCACGGAAGTT
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,
, All fractions were stored at -80°C until further use. Secretomes were collected, clarified and stored. Cell monolayers were washed twice in PBS, scrapped in 1 mL PBS-Triton X-100 1%, transferred to Eppendorf tubes and centrifuged. The soluble fraction was collected and its protein content was measured using Pierce® BCA Protein Assay Kit (23225 Thermo Scientific) as recommended by manufacturer's instructions. The insoluble fraction was washed once in 1 mL PBS-Triton X-100 1%, p.500
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