Tgf β2

AGC1^+/+ and AGC1^+/− SVZ-derived neurospheres were counted and plated on 13-mm coverslips (50 neurospheres/well) previously treated with poly-l-lysine (10 µg/mL) and then fibronectin (1 µg in complete DMEM-F12 medium with 10 ng/mL TGFβ2 (Sigma-Aldrich) for 4 days in an incubator at 37 °C. After 4 days, neurospheres were fixed with 4% PFA for 30 min, washed with PBS and stored at 4 °C in PBS until used for immunofluorescence analysis or resuspended in cell lysis buffer (50 mM Tris pH 7.4, 1 mM EDTA, 1% SDS, 10 µL/mL protease and phosphatase inhibitor cocktails) for western blot analysis.

Chloroquine (CQ), 3-methyladenine (3-MA), rapamycin and TGF- β₂ were purchased from Sigma-Aldrich (St Louis, MO, USA). LY2109761 was from Selleck Chemicals (Houston, TX, USA). Antibodies against microtubule-associated protein 1 light chain 3-I (LC3-I), LC3-II, p62, Beclin 1, ATG7, poly (ADP-ribose) polymerase (PARP), cleaved-PARP, caspase 9, cleaved-caspase 9, Bax, and Bcl-x1 were from Abcam (Cambridge, MA, USA). Antibodies against TGF-β₂, Smad2, p-Smad2, E-cadherin, α-catenin, β-catenin, N-cadherin, fibronectin, ZO-1, Vimentin, MMP-9, Snail, Slug and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies of LC3-II and E-cadherin and secondary antibodies Alexa Fluor 568 anti-mouse IgG and Alexa Flour 568 anti-rabbit antibodies were from Jackson Immuno Research (Lancaster, PA, USA).

Human TM cells between passages 5 and 7 were grown to 80% confluence and growth arrested using serum free medium prior to stimulation. Cells were stimulated with recombinant human TGF-β2 (R&D Systems, UK) at a concentration of 5 ng/mL for 24 h. Vehicle control cells were stimulated with equal volumes of 4 mM HCl and 0.1% BSA solution (Sigma, UK). The viability of TM cells treated with TGF-β2 was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT, Sigma-Aldrich, USA) (Supplemental Fig. 1C). 24 h post TGF-β2 treatment, 10uM of MTT solution was added to each well and incubated for 3 h. After incubation, the media was removed and the formazan crystals were dissolved in 100 µl of DMSO. The optical densities (OD) of the dissolved formazan crystals was read on a plate reader at 570 nm (Omega FluroStar; US). The quantification of cell viability was obtained by comparing the optical density of the treated and untreated samples. The relative cell viability was calculated for each tissue as Arbitral Unit (AU), extrapolated by Optical Density (OD) of the samples.

Smad3 (phos-S208) (Abcam, ab138659), SERPINE1 (Fisher Scientific), Rap 1 (sc-398755) and TGF-β RI Kinase inhibitor V (Santa Cruz Biotechnology), PARP (#9542S), LC3 A/B (#4108S), ZYX, and total Smad3 (Cell Signaling Technology) antibodies were obtained from commercial vendors. Simvastatin was obtained from Sigma Chemical (in vitro stock and in vivo stock for subcutaneous GIC grafts) and Selleckchem (in vivo stock for GIC intracranially grafted mice). TGF-β2, mevastatin, fluvastatin, lovastatin, geranylgeranyl pyrophosphate (GGPP), farnesyl pyrophosphate (FPP) and (±)-mevalonolactone were obtained from Sigma Chemical. Silencer® select pre-designed SMAD3 siRNAs, and Silencer® Select Negative Control siRNA were purchased from Life Technology. LY2109761 was from AdooQ BioScience (A11133).

Cellular total RNA were extracted from WT and lenti TGFBRII-gRNA/Cas9-treated cells using Quick-RNA MiniPrep Kit (ZYMO Research, Irvine, CA), respectively. One μg of RNA were subjected to reverse transcription using SuperScript^® VILO^™ MasterMix (Invitrogen Corporation), followed by qPCR, using iTaq^® Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA) on Applied Biosystem^® 7900HT Fast Real-Time PCR System (Life Technologies), for primers: BMP-7, CD133, EpCAM, FOXC2, FZD4, GSK3B, MMP9, SNAIL2, SOX9, TCF3, TGF-β2, TGF- β3, TWIST1, WNT5A, WNT5B, ZEB1, ZEB2, and GAPDH (all from Sigma-Aldrich Co. LLC.; Supplementary Table S1) [6 (link)].

Oli-Neu cells were plated at the density of 1 × 10⁵ in 6-well plates containing poly-l-lysine (10 µg/mL) coated glass coverslips. Cells were allowed to adhere for 2 h before adding 10 ng/mL TGFβ2 (final concentration; Sigma-Aldrich). After 24 h, cells were fixed for 30 min at RT with 4% PFA in phosphate buffer (0.194 M Na₂HPO₄, 0.026 M NaH₂PO₄) and stored in PBS at 4°C until used for cell filament measurement and immunofluorescence or resuspended in cell lysis buffer (50 mM Tris pH 7.4, 1 mM EDTA, 1% SDS, 10 µL/mL protease and phosphatase inhibitor cocktails) for western blot analysis.