Olig2

OPCs were plated on 8‐well Permanox chamber‐slides (Nunc, Naperville, IL), precoated with 5 µg/mL PLL followed by appropriate aFn or pFn coatings (described above), at a density of 30,000 cells per well. OPCs were allowed to incorporate 5‐bromo‐2‐deoxyuridine (BrdU; 10 µM; Roche) for 24 h in the presence of 10 ng/mL PDGF‐AA and 10 ng/mL FGF‐2. Then, cells were fixed in 4% PFA for 20 min, and additionally fixed in 5% acetic acid in ethanol for 20 min. BrdU was detected using reagents from the BrdU labeling and Detection Kit I (Roche) according to the manufacturer's instructions with the addition of the oligodendrocyte lineage marker Olig2 (Abcam) and Alexa Fluor© 546‐conjugated anti‐rabbit antibody, and visualization of nuclei with DAPI (1 µg/mL). To compare the percentages of BrdU‐incorporating cells between the conditions, the numbers of double BrdU‐ and Olig2‐positive cells were counted relative to the Olig2‐positive cells (at least 150 cells per condition) from images captured with a Leica TCS SP8 Confocal Laser Scanning Microscope.

After OSM and STAT3 inhibitor treatment, the total proteins were extracted by RIPA buffer (Pierce, Rockford, IL) on ice. Equal quantities of protein samples were separated by electrophoresis on 12% SDS-PAGE polyacrylamide gels. Then, the samples were electro-transferred to PVDF membranes (0.22 μm, Millipore) using a wet transfer apparatus (Bio‐Rad) and blocked with 5% BSA in PBS for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies of CD44, FN1, CHI3L1, CD24, DLL3, OLIG2 and β-ACTIN (Abcam), respectively. After that, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#HA1001, huabio, 1:5000) at room temperature for 1 h [30 (link)]. The immobilon reagents (Millipore) was used for the visualization and detection of antibody-antigen complexes. The band intensity was measured by ImageJ software.

Following treatment, coverslips were gently washed with PBS before fixation with 4% PFA. Following fixation, cells were permeabilized using 5% goat serum (Thermo Fisher Scientific, MA, USA) and 0.01% Tween20 (Sigma-Aldrich, MA, USA). Cells were stained using 4’6-diamidino-2-phenylindole (DAPI) to identify nuclei, as well as the indicated primary antibodies, including MBP (1:500; Abcam, Cambridge, UK), Olig2 (1:500; Abcam, Cambridge, UK), and NFκB (1:200; Thermo Fisher Scientific, MA, USA). Conjugated secondary antisera directed against the species of the primary were used according to manufacturer instructions (1:1,000, Abcam, Cambridge, UK). Coverslips were then affixed to slides (Denville Ultraclear, MA, USA) with fluromount-G (Invitrogen, MA, USA) and imaged (Olympus 1X71, CellSens Software; Olympus, MA, USA). Five fields of view at 20× magnification using identical image capture settings were assessed by an experimenter blinded to treatments. To analyze the amount of OPC differentiation, Olig2+ cells and MBP+ cells were counted and the percent MBP+ cells was calculated. Olig2+ was also used to distinguish astrocytes in the cultures to refine the cell counts to only MBP+/Olig2+ OL-lineage cells. Data are presented as the percentage of MBP+/Olig2+ cells relative to the control, differentiation media only, condition set as 100%.

The Olig2 protein is transcription factor known to be required for proliferation of glial tumors and regulated by several sialoglycan expression [27 (link), 28 (link)]. Naïve and Dex-treated cells were scraped, diluted to 10⁵ per sample and incubated with Olig2 (Abcam, 2,5 µg/ml) antibody for 30 min at 4 °C. To facilitate intracellular staining, 0.01% Triton was used. Cells were washed with phosphate buffered saline, stained with appropriate secondary fluorescent antibody and analysed on Becton Dickinson flow cytometry system. In each analysis, corresponding isotype control antibody was used as a negative control.

Brainstem slices (100 μm thick) were cut and subsequently fixed with 4% (wt/vol) paraformaldehyde in phosphate buffer solution (PBS) for 20 min. Free‐floating sections were blocked in 4% goat serum and 0.3% Triton X‐100 in PBS for 1 hr. Slices were incubated with antibodies for chicken anti‐green fluorescent protein (GFP; 1:300; Abcam, Cambridge, UK, Cat# ab13970, RRID:AB_300798), rabbit anti‐oligodendrocyte transcription factor 2 (Olig2; 1:200; Abcam, Cambridge, UK, Cat# ab109186, RRID:AB_10861310), mouse anti‐neuronal nuclei (NeuN; 1:600; Millipore, Burlington, MA, USA, Cat# MAB377, RRID:AB_2298772) overnight at 4°C and subsequently incubated with different Alexa dye–conjugated secondary antibodies (1:500, Invitrogen, Carlsbad, CA, USA) for 2 hr at room temperature. Slices were mounted onto Superfrost slides in photobleaching‐protective medium (Fluoroshield; Sigma‐Aldrich, St. Louis, MO, USA). Stained slices were viewed with laser lines at 488, 555, and 633 nm using a 40× oil‐immersion objective on a confocal laser‐scanning microscope (LSM‐510, Zeiss, Oberkochen, Germany). Stack images were acquired at a digital size of 1,024 × 1,024 pixels with optical section separation (z interval) of 0.5 μm and were later cropped to the relevant part of the field without changing the resolution.