Fig 2: TLX is essential for tumor sphere formation. (a) Representative images of monolayer (contains serum) and IMR-32 spheres (serum-free). Bar, 20 μm. Lower panel depicts representative images obtained by sphere formation assays. IMR-32 WT, ShCtrl, Sh2 and Sh3 cells were cultured for 2–3 weeks in the defined media for sphere formation and spheres collected and counted after indicated time intervals. (b) Quantitation of the number of spheres after indicated time intervals in control or TLX-silenced cells. (c) Number of spheres per 1000 cells derived from primary spheres in subsphere formation assay. (d) Immunoblot analysis of monolayer (Mon), primary (Pri) or primary-derived secondary spheres (Sec) of IMR-32 cells for TLX expression. GAPDH is used as loading control. (e) Immunofluorescence image of IMR-32 spheroid double stained for CD133 and TLX (bar, 100 μm) and the larger magnification (bar, 20 μm). (f) TLX transcript levels were measured by qPCR and normalized to GAPDH in CD133-positive and -negative cells derived from from single-cell suspension of spheroids sorted using CD133 Microbead Kit (Miltenyi Biotec). Control set to 1±S.D.

Fig 3: TLX transcriptionally regulates MMP-2 and Oct-4 in hypoxic NB cells. (a) Luciferase activity in 293T cells after co-transfection of MMP-2 promoter-luciferase constructs with TLX or control vector. (b and c) Top panels depict schematic representation of regions analyzed by ChIP within MMP-2 promoter or Oct-4 promoter (c). Occupancy of TLX, Pol-II and H3K9 acetylation across the 1.2 kb upstream regulatory regions of TLX-regulated genes MMP-2 and OCT-4, and control actin promoter was monitored by ChIP analysis upon normoxia (b) or hypoxia (c). Chromatin was isolated from normoxia- or hypoxia-treated cells and ChIP analysis was performed as described in Materials and Methods. Amplicon from each immunoprecipitate is represented as the percentage of input. Each error bar indicates standard deviation calculated from triplicates. (d) Graph represents the binding of TLX to MMP-2 promoter as a function of absorbance at 450/650 nm. Biotin-labeled consensus oligos were used to capture TLX of nuclear lysate from WT IMR-32. A nonspecific capture oligo served as control, and rabbit IgG were used to exclude nonspecific binding. Mutant oligos (Mut1 or Mut2) were used to confirm the specificity of capture. The values obtained are means of three independent experiments along with S.D. as error bars

Fig 4: TLX is enriched in proliferative cells of spheres. (a) Representative images of monolayers and spheroids from IMR-32, LAN-5 and SK-N-BE2c cells. The numbers below indicate the percentage of TLX- and CD133- expressing cells analyzed by FACS. (b) Dispersed SK-N-BE2c spheres stained for TLX (red) and indicated proteins (green) (right panel). Images were obtained by confocal microscopy. Bar, 10 μm. (c) Single-cell suspension of spheres were differentiated using 1% FBS and cells were stained for coexpression of TLX using differentiation markers GFAP and MAP2ab

Fig 5: TLX expression is elevated in NB cell lines. (a) Immunoblot analysis for TLX in equal amounts of protein from cell extracts of NB cells lines, namely SH-SY5Y, SK-N-SH, SK-N-BE2c, LAN-5 and IMR-32. GAPDH was included as a loading control. (b) Immunoblot analysis of TLX in shRNA-control (ShCtrl) or TLX-specific shRNA (Sh2, Sh3)-derived stable clones. (c) Phase-contrast image of IMR-32 WT, control and ShTLX cultured under normal proliferation conditions. Magnification, × 20. (d) Fold expansion of viable cells at different time points at 24, 48 and 72 h of IMR-32, ShCtrl, Sh2 and Sh3. (e) Immunoblot analysis for TLX, P-Akt and total-Akt (T-Akt) in equal amount of proteins from the extracts of IMR-32 WT, Si Ctrl and transiently silenced TLX Si1 and Si2 cell lines. GAPDH was used as a loading control