Figure 3

LiCl treatment decreased MB tumor sphere proliferation and increased senescence. (a) Primary Ptch^+/- MB cells on coverslips (3×10⁵cells/well) were treated for 72 h with 10 mM NaCl or LiCl and stained with rat-anti-Ki67 (DakoCytomation), Alexa-Fluor-568 goat-anti-rat (life technologies) and DAPI (Sigma-Aldrich). Ki67-positive cells were counted (Nikon Eclipse 80i; 20×; NA 0.5), normalized to total cell counts (n = 4, mean ± s.d., p-values *** ˂0.0001) and representative pictures were taken by confocal microscopy (Nikon Eclipse C1si; 40×/60×, NA 1.3/1.4; oil). Scale ≙ 50 μm. (b) Ptch^+/- cells were seeded on μ-Slides/ibi-Treat (ibidi) (8×10⁴cells/well), treated with 10 mM NaCl or LiCl and stained with rabbit-anti-pHH3 (Ser10, Merck Millipore), Alexa-Fluor-568 goat-anti-rabbit (life technologies) and DAPI. pHH3-Positive cells were visualized by confocal microscopy (40×, NA 1.3; oil), counted and normalized to total cell counts. Bars represent mean ± s.d., (n = 4, p-value *0.0201). Scale ≙ 50 μm. (c) Cell cycle was evaluated by propidiumjodid (5×10⁵ cells; PI, Sigma-Aldrich), measured with a FACS Canto II (Becton Dickinson) and analysed with ModFit LT 3.2 (Verity Software House). Bars represent cell distribution in G2/M, S and G1 phase after 24 h or 72 h of NaCl or LiCl treatment (each 10 mM). (d) Bars represent cells in G2/M phase after NaCl or LiCl treatment (mean ± s.d.; n = 3, p-value *0.0147). (e) LiCl increased SA-βGal-positive Ptch^+/- MB cells after 72 h, counted in high power field (40×/0.55NA; Nikon Eclipse TS100). Bars represent mean ± s.d. (n = 3, p-value **0.0045). (f) Increased senescence of Ptch^+/- cells by LiCl associated with increased p21 mRNA levels (see Table 1 for primers). Axin2 served as a control for β-catenin transcription. Dashed line represents NaCl control (n = 6, p values left to right: *0.0150, *0.0046; Unpaired student’s t-test in GraphPad Prism 5.01).