Figure 5

LiCl treatment delayed growth and decreased volume of Ptch ^+/- MB-derived tumors. (a) 1×10⁶ cells in 100 μl PBS were subcutaneously injected into HsdCpb:NMRI-Foxn1nu mice (6-8weeks, Harlan Laboratories) and animals were treated with LiCl (7,5 μl/g body weight, 1.2 M) or NaCl (7,5 μl/g body weight, 1.2 M) from day 5 post-transplantation. From day 21 tumors were measured daily thereafter (electronic disk micrometers, Vogel Germany), the mean of tumor volume was determined by the formula: π/6× maximal-diameter x minimal-diameter² (mm³) until animals were sacrificed by day 32. (b) Dots represent the percentage of tumor-bearing animals. Statics were performed using PSPP 0.8.2 as described previously [23]. Equal numbers (n = 7) from NaCl- or LiCl-treated mice were tested for tumor formation probability by a non-parametric one-tailed binomial proportions test, setting the expected outcome for tumor formation to 90%. There was no significant difference between the expectation and the tumor incidence for the NaCl-treated group (p-value 0.52), but for the LiCl-treated group (p-value *0.03). (c) Paraffin sections (3 μm) of tumors were stained with hematoxylin and eosin, mitotic figures were counted per high power field and pictures were taken at an Olympus BX50 microscope 40× (NA 0.75). Bars represent mean ± s.d. (n = 7 (NaCl), n = 6 (LiCl)), p-value **0.0021). Scale bar ≙ 50 μm. Analysis was done with GraphPad Prism 5.01. (d) Immunohistochemistry with anti-pHH3 (Merck Millipore) antibody on paraffin sections (3 μm) was performed using Ventana Discovery XT system (Ventana, USA), examined with the Axiophot (Zeiss, Germany, Achroplan 0.65) and analyzed in Stereo Investigator 4.34 (MicroBright Field.Inc Europe, Germany). Pictures were taken at a Nikon Eclipse 80i (Nikon, Japan; 10×/40×; NA 0.5). Bars represent mean ± s.d. (n = 7 (NaCl), n = 6 (LiCl), p-value *0.0463). Scale bar represent 200 μm (left), 50 μm (right).