Fig. 4

Gene expression changes, reduced invasion, and CalR translocation after MitA mono- and combination therapy. A Quantitative qPCR was conducted with cDNA from control cells and after treatment upon reverse transcription of total RNA as described in material and methods. Analysis was done in triplicates with n = 5 independent experiments. All data are given as 2^−ΔΔCT values + SD. *p < 0.05, **p < 0.01 vs. control, #p < 0.05 vs. control; ***p < 0.01 vs. control; ****p < 0.0001 vs. control; #p < 0.05 vs. monotherapy, ##p < 0.01 vs. monotherapy ###p < 0.001 vs. monotherapy, ####p < 0.0001 vs. monotherapy, Two-way ANOVA (Tukey’s multiple comparisons test). B Effects of MitA and its combination on cell invasion (HROG05 [black], HROG63 [grey]) assessed by a Boyden chamber assay [absorbance: 450 nm]. Mean + SEM, n = 3 independent experiments. *p < 0.05, **p < 0.01 vs. control, One-way ANOVA (Tukey’s multiple comparison test). C Representative images of MitA-treated 3D-cultured GBM cell invasion into a Matrigel matrix (scale bar: 250 μm). GBM spheres were monitored for a total of 10 days and images were taken on day 0, 5 and 10 using the Leica microscope DMI 4000B. Representative images of n = 3 independent experiments. D CalR translocation was detected after 2 × 72 h mono- and SIM combination treatment by staining CalR on the cell surface. Upper part: representative dots plots showing CalR-positive cells. Lower part: Quantitative analysis. 10,000 events were measured and the percentage [%] of cells showing CalR translocation were provided. n = 3 independent experiments. *p < 0.05, **p < 0.01 vs. control, One-way ANOVA