Supplementary data from The Novel Tubulin-Binding Checkpoint Activator BAL101553 Inhibits EB1-Dependent Migration and Invasion and Promotes Differentiation of Glioblastoma Stem-like Cells

Materials and methods: EB1 Western blot analysis GBM stem cells were lysed (lysis buffer: Tris 50 mM pH 8.0, NaCl 250 mM, Triton-X100 1%, SDS 0.1% with a cocktail of proteases and phosphatases inhibitors added freshly (all from Sigma-Aldrich). Thirty µg of total protein lysate were loaded onto a 12% SDS-PAGE gel. Nitrocellulose membrane (Bio-Rad laboratories, Marnes la Coquette, France) was blocked with 5% milk (powder) in Phosphate Buffer Saline (PBS) (Life technologies)-Tween (Sigma-Aldrich) pH 7.4 for 1 h and then incubated in PBS-Tween 0.1%-5% milk solution with mouse anti-EB1 antibody (clone 5, BD Biosciences, Le pont de Claix, France) (1/1000) and mouse α-tubulin (clone DM1A, Sigma Aldrich). After washing, membranes were incubated with anti-mouse peroxydase-conjugated secondary antibodies (Jackson Immunoresearch, Baltimore, USA) for 1 h. The bound antibodies were then detected using chemiluminescence detection kit (Millipore, Saint Quentin en Yvelines, France). Signals were recorded with G:BOX (Syngene/Ozyme, Saint Quentin en Yvelines, France ) and quantification was done with Image J software. Immunofluorescence analysis Indirect immunofluorescence was performed as previously described (15). Briefly, cells were fixed for 5 min with cold methanol (-20{degree sign}C), incubated with the anti-EB1 antibody 1/100 for 1 h and anti-mouse antibody Alexa 568 nm (Molecular Probes) 1/200; and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich) 1/200 for 1 h at room temperature in the presence of DAPI for nuclear staining. For GFAP staining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 for 10 minutes. Primary antibody against GFAP (rabbit IgG, 10€‰Î¼g/ml, Dako) was incubated overnight at 4€‰{degree sign}C. Secondary antibody, Texas-red Goat anti-rabbit IgG (H+L) was purchased from Jackson Immunoresearch (Newmarket, UK), and was incubated for 1 h. Cells were analyzed using a Leica DM-IRBE microscope at a magnification of x100. Real-time quantitative PCR RNA samples were processed using a LightCycler 480 instrument (Roche Applied Science) and a LightCycler 480 SYBR Green I Master Mix (Roche Applied Science). Briefly, total DNA-free RNA (1 μg) was reverse-transcribed into cDNA using 1 μg of random hexamers and Superscript II reverse transcriptase as recommended by the manufacturer (Invitrogen Life Technologies, Cergy Pontoise, France). Measurements were performed in triplicate for each sample, and relative expression ratios of target gene transcripts (GFAP, TUBB3, CNP) and reference gene transcripts (18S, GAPDH, and ACTB) were calculated using qPCR efficiencies and cycle threshold (Ct) deviations of tumor and normal adult brain samples (control: Agilent Technologies) (25). RNA expression levels in GBM CSLCs were subsequently expressed as percentages compared to normal adult human brain samples corresponding to 100% of expression. Forward and reverse primers used for each gene are listed in Supplemental Table 1. Supplementary Table 1: Sequences of primers used in RT-qPCR Supplementary Figure 1: Chemical structure of BAL27862 and BAL101553 Supplementary Figure 2: Modulation of EB1 expression in GBM6 cells using siRNA or shRNA against EB1. (A) Western blot analysis of EB1 in GBM6 shEB1 clones (GBM6 shEB1, GBM6 shEB1#2), in GBM6 GFP shEB1 clone and in control clones (GBM6 wt, GBM6 sh0 and GBM6 GFP sh0). Ratios EB1/tubulin, relative to controls, from at least three independent experiments are presented under the blots. (B) Analysis of EB1 expression by Western blot of GBM6 wt or treated with siRNA against EB1 (GBM6 siEB1) or siRNA control (GBM6 si0). (C) Immunofluorescence staining of tubulin (green) and EB1 (red) in GBM6 wt, GBM6 si0 and GBM6 siEB1. Bar = 10 μm.