Af6000 modular microscope

The activated PKA protein, p-PKA, was visualized by performing immunofluorescence experiments. A total of 10 × 10³ cells per well were seeded in 96-well plates and treated for 20 min, 1 h, 24 h, and 48 h in presence of HPE_DMSO. In order to analyze the effects of HPE_DMSO under forskolin stimulation, the cells were treated with forskolin for 30 min or pre-treated with HPE_DMSO for 1 h and then stimulated with forskolin for 30 min. At the end of treatments, cells were washed in PBS, fixed in 100% ethanol for 15 min, and permeabilized with 0.5% Triton-X 100 in PBS for 10 min. After blocking with 3% bovine serum albumin (BSA) in PBS for 30 min, cells were incubated at 1 h with rabbit oligoclonal anti-p-PKA antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) 1:250. Cells were washed with PBS and then incubated for 1 h with Alexa Fluor 595 donkey anti-rabbit antibody 1:400 (Invitrogen, Thermo Fisher Scientific, to stain p-PKA in red. All steps were performed at room temperature. Slides were washed and then stained with DAPI (Invitrogen, Thermo Fisher Scientific) to visualize the nuclei. The images were captured by optical microscope Leica DM IL LED, using AF6000 modular Microscope (Leica Microsystem, Milan, Italy).

Collagen-II protein was visualized by immunofluorescence. HPCs were plated at a density of 8 × 10³/cm², and left untreated (CTL) or treated, for 72 h with different concentrations of GlcNAc and GlcNAc NP. Cells were washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min at 4 °C, and permeabilized with 0.5% Triton-X 100 in PBS for 10 min at room temperature (RT). Then, cellular proteins were blocked with 3% bovine serum albumin in PBS for 30 min, and cells were incubated for 1 h with anti-Collagen-II (1:100) primary antibody. Cells were washed with PBS and then incubated with Alexa Fluor 595 donkey anti-mouse red secondary antibody (1:400) for 1 h. Cells were washed and then stained with DAPI to visualise the nuclei. All these steps were performed at RT. The images were captured by a Leica DM IL LED optical microscope, using an AF6000 modular microscope (Leica Microsystem, Milan, Italy).

Vimentin, CB2, and PI-PLC β2 were visualized by immunofluorescence. Cells were plated at a density of 8 × 10³/cm² and cultured for 48 h and then, washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min at 4 °C, and permeabilized with 0.5% Triton-X 100 in PBS for 10 min at room temperature. After blocking with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature, cells were incubated at 1 h, at room temperature, with mouse monoclonal anti-vimentin antibody (Proteintech Group, Manchester, UK) 1:50, mouse monoclonal anti-CB2 antibody 1:150, and mouse monoclonal anti-PI-PLC β2 antibody (Santa Cruz Biotechnology) 1:50. Cells were washed with PBS and then, incubated for 1 h, at room temperature, with Alexa Fluor 488 donkey anti-rabbit antibody 1:300 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), to stain vimentin green; with Alexa Fluor 488 donkey anti-goat antibody 1:600 (Invitrogen, Thermo Fisher Scientific), to stain CB2 receptors green; and Alexa Fluor 595 donkey anti-rabbit antibody 1:300 (Invitrogen, Thermo Fisher Scientific), to stain PI-PLC β2 red. Slides were washed and then, stained with DAPI (Invitrogen, Thermo Fisher Scientific) to visualize the nuclei. The images were captured by a Leica DM IL LED optical microscope, using an AF6000 modular microscope (Leica Microsystem, Milan, Italy).