Nestin

For immunofluorescence staining, mNPCs were plated on the coverslips and fixed using 4% paraformaldehyde (PFA) for 20 min, then permeabilized with 0.4% Triton-X in PBS for 15 min. Subsequently, the coverslips were blocked with1% BSA for 1 h. mNPCs were incubated with primary antibodies overnight including Nestin (1:500; Novus Biologicals) and Ki67 (1:1,000; Cell Signaling Technologies). Coverslips were washed with PBS 3 times and incubated for 1 h at room temperature with secondary antibodies including anti-rabbit, mouse or chicken IgG (coupled with Alexa Fluor 488 or 568, Life Technologies). Nuclei were counter-stained with DAPI. Coverglasses were fixed on glass slides with Mounting Medium (Sigma-Aldrich). The images were captured by Zeiss AX10 fluorescence microscope. For quantification, the numbers of stained cells were quantified by Image-Pro Plus 6.0.

Lysates of GBM TSs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on Tris–glycine gels. Cytosolic and nuclear fractions were prepared using cytoplasmic and nuclear extraction reagents (Thermo Scientific, Rockford, IL, USA), respectively, according to the manufacturer’s instructions. Proteins were transferred to nitrocellulose membranes and probed with antibodies against cleaved capase-3, CD133, PDPN, β-catenin, active-β-catenin, CD44, snail, Cyclin D1 and cMYC (Cell Signaling Technology, Danvers, MA, USA); Sox2 (Merck Millipore, Darmstadt, Germany); Msi-1 (Abcam, Cambridge, UK); nestin (Novus Biologicals, Centennial, CO, USA); N-cadherin, Zeb1 (Sigma-Aldrich, St. Louis, MO, USA); Twist, Oct3/4, BAX, Bcl-2, PARP and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA); and FOXM1 (Bethyl Laboratories, Montgomery, TX, USA), along with Western Lightning Plus-enhanced chemiluminescence reagent (PerkinElmer, Lawrence, MA, USA). Images were captured using an ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Little Chalfont, UK). We expressed all the results of Western blot as a violin plot, and added this as a Supplementary 5. The original image of full-length blot was added as a Supplementary 6.

For cytofluorimetric analysis adherent cells were trypsinized, then washed twice in PBS, incubated with 5 μl of fluorochrome-conjugated antibodies for 30 min at 4 °C (5 × 10⁵cells/FACS tube), fixed with 1% formaldehyde solutions for 5 min at 4 °C, centrifuged for 5 min at 1600 rpm and washed once with 1 ml of PBS for 5 min at 1600 rpm. CD133/2 (293C3) (Miltenyi Biotec, Bergisch Gladbach, Germany), HLA-I, NGF-R, ICAM-1, CD20, CXCR4, CD10, c-Kit antibodies (all from BD Biosciences, Franklin Lakes, NJ, USA) conjugated with different fluorescent dyes were used; the unconjugated antibodies Nestin (Novus Biologicals, Minneapolis, USA) and Melan-A/MART-1 (Santa Cruz Biotechnology, Dallas, TX, USA) were used in combination of the goat-anti-mouse IgG-FITC (BD Biosciences) as secondary antibody and used after cell permeabilization. For apoptosis analysis cells were trypsinized, washed in PBS, fixed with 70% ethanol for 45 min at 4 °C, washed in PBS and stained with propidium iodide (50 μg/ml diluted in PBS) and RNAase (250 μg/ml), then stored for at least 3 h at 4 °C before analysis. Flow cytometer analysis was performed by BD FACScan™ System using CellQuest Pro software on a minimum of 5000 events for each sample.

The spinal cord tissues were fixed in 10% formaldehyde, embedded in paraffin and cut into 5‐μm‐thick transverse sections. Hematoxylin and eosin (H&E) staining and Nissl staining were performed according to the manufacturer's instructions. For immunofluorescence, 10‐μm‐thick transverse frozen sections were incubated with primary antibodies targeting MAP‐2 (1:200) and Nrf2 (1:200, R&D systems), and the longitudinal sections, with antibodies against GFAP (1:500, Abcam), NF‐200 (1:200, Boster), Tuj1 (1:200, CST), GAP43 (1:300, Novus), BrdU (1:200), Nestin (1:200, Novus), NeuN (1:300) and NSE (1:100, Abcam). After incubation with species‐specific secondary antibodies conjugated with Alexa Fluor 488 (1:300, CST) or Cyanine3 (1:200, Invitrogen) for 1 hour at room temperature, the sections were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and observed under a laser‐scanning confocal microscope (Leica).

Neuronal cells, hiPSC-derived organoids, and mouse brain slices mounted on glass slides, were fixed with 4% paraformaldehyde. Samples were blocked for 1 hour with 2.5% BSA before incubating with primary antibodies at 4°C overnight. Primary antibodies used for IF were phospho-tau-AT8 (#MN1020, ThermoFisher), anti-tau-1 (#MAB3420, Millipore Sigma), eEF2, eEF2K, β-tubulin (#2332, #3692S, #2146S, Cell Signaling), Nestin (#NB100–1604, Novus). Samples were washed with PBS containing 0.1% TritonX-100 (PBST) prior to incubation with AlexaFluor secondary antibodies for 1 hour at room temperature. Images were captured on Cytation 5.0 under identical capture settings. High resolution images (60X silicon) were captured using the Andor Spinning-Disk Confocal microscope. Image analyses were conducted using QuPath open source software.

Cells were cultured on Lab‐Tek chamber slides (Nalgene Nunc International) until 40% confluent before treatment with either TGF‐β1 or vehicle control, at the indicated time points. Cells were fixed with 4% formaldehyde and processed for immunofluorescence microscopy (Kyrkanides et al., 2012 (link)). Primary antibodies included Oct4 and Nanog (Abcam), Nestin (Novus Biologicals), counter stained with DAPI (Vector Laboratories). Images of cover slipped slides were captured under 594 nm (red) and 350 nm (blue) wavelengths using a BX51 Olympus fluorescent microscope.

Samples were prepared as described previously (Al‐Attar et al., 2018 (link)). Briefly, harvested cells were fixed with 2% paraformaldehyde, washed with permeabilization buffer (BioLegend) and incubated with primary antibodies: Oct4 (Chemicon), Nanog (Cell Signaling), Sox2 (Santa Cruz Biotechnology), Nestin (Novus Biologicals), and Rabbit IgG (Abcam) and chicken IgY isotype (R&D Systems) controls. After rinsing, cells were incubated with an Alexa flour‐488 goat anti‐rabbit secondary antibody (Molecular Probes), or a FITC goat anti‐chicken IgY (Novus Biologicals) isotype control. Stained samples were read on an LSR‐II cytometer using FACSDiva version 6 (BD Biosciences) and analyzed by FlowJo Software (TreeStar; version 9.5). Data are represented as geometric mean‐fluorescence intensity (GMFI).