Alexa fluor 680

For protein extract preparation, cells were lysed on ice with RIPA Lysis Buffer (ThermoFisher) containing complete protease and phosphatase inhibitor cocktail (Roche). Soluble protein extracts were separated by centrifugation at 13000 rpm for 15 min and diluted in Laemlli sample buffer. The obtained cell lysates were resolved on sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and transferred on polyvinylidene difluoride membrane Hybond TM-P (Amersham Bioscience). Membranes were saturated with 5% bovine serum albumin at room temperature for 2 h and incubated with the following primary antibodies at 4 °C overnight. Primary antibodies were used as follows: ELF3 (1:1000, AF5787; R&D Systems, Minneapolis, USA), EHF (1:1000, 27195-1-AP; Proteintech, Illinois, USA), TGIF1 (1:1000, ab52955; Abcam, Masseachusettes, USA), β-actin (1:1000, 3700S; CST, Boston, USA) and GAPDH (1:1000, 2118S; CST, Boston, USA). Secondary anti-mouse IgG (ab175775), anti-rabbit IgG (ab175773), and anti-goat IgG (ab175776) all conjugated to Alexa Fluor 680 (Abcam, Masseachusettes, USA) were incubated with the membranes for 2 h at room temperature at 1:10,000 dilution. All bands of western blot were detected and qualified with gray scale ratio by Odyssey CLx imaging systems (LI-COR, Nebraska, USA).

Tissue or neurons were lysed in 50 mM Tris-HCl pH7.6, 150 mM NaCl, 2 mM EDTA, 2% SDS or RIPA buffer (Sigma Aldrich) supplemented with complete protease inhibitor mixture (Roche Applied Biosciences) and phosphatase inhibitor mixture (Pierce Biotechnology). Proteins were separated by 4–12% NuPage Bis-Tris PAGE (Invitrogen) and transferred to nitrocellulose membranes. Antibodies specific for MOR1 (1:1000, Millipore), DARPP32-pT75 (1:1000, Cell Signaling), DARPP32-pT34 (1:1000, Cell Signaling), DARPP-32 (1:1000, Cell Signaling), ALDH1A1 (1:1000, Sigma-Aldrich), pERK (1:1000, Cell Signaling), ERK (1:1000, Cell Signaling) were used. Anti-GAPDH antibody (1:1000, Sigma–Aldrich), Anti-β-actin antibody (1:1000, Sigma–Aldrich) were used to adjust equal amounts of protein loading into each well. The secondary antibodies conjugated with Alexa Fluor® 680 or Alexa Fluor® 800 were purchased from Abcam. Protein bands were detected using Odyssey CLx Imaging System (LI-COR) and quantified using the Scion Image System.

The C-20/A4 chondrocytes were seeded with an approximate density of 263,000 cells/cm² in sterile 12-well culture plates on the coverslips pre-coated with poly-l-lysine. After treatment with 200 or 300 µM FAC for 24 and 48 h, the cells were washed with PBS and fixed with paraformaldehyde (4%) at room temperature for 15 min. Post fixation cells were permeabilised for 10 min by treatment with 0.1% Triton X-100. Later, cells were blocked with 3% BSA for 1 h, rinsed with 1X PBS, and incubated at 4°C overnight with primary antibodies diluted at 1:500. The primary antibodies used were anti-Collagen II antibodies: (Cat No. ab185430) from LifeSpan Biosciences, Seattle, Washington. Cells were then washed with 1X PBS and incubated for an hour at 37°C with Alexafluor^®680-labelled (Abcam) secondary antibodies, rinsed with 1X PBS. To stain the nuclei, mounting medium with ProLong gold antifade mountant with 4′,6′- diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, United States) was used. The cells were visualised by using an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan). The images were analysed for quantification of collagen II levels by using ImageJ software (Schneider et al., 2012 (link)).