Fig. 5

Stable overexpression of native SGPL1. Evaluation of native SGPL1 restauration effects on SGPL1 and SPHK1/2 isoenzyme transcript and protein expression level as well as on cell physiology and SGPL1 localization, representatively in transfected RH-30 cells. The expression factors were all determined densitometrically and normalized to the mock transfection control (C.), which was set to 1. (legend: n.t. = non-transfection control, C. = mock transfection control, SGPL1 = SGPL1 restauration, siRNA+ = positive siRNA control, siRNA− = negative siRNA control and ΔSGPL1 = SGPL1 siRNA). a Transcript expression analysis by RT-PCR of SGPL1, SPHK1, SPHK2 as well as controls: β-actin and GAPDH (loading control); PAX3-FOXO1 (RMA marker); as well as ezrin and CXCR4 (metastasis marker). (Representative images of three independent experiments, n = 3). b Western blot images of SGPL1, SPHK1, SPHK2 and β-actin (loading control) as well as proliferation marker PCNA and anti-apoptotic protein marker Bcl-2 (Representative images of three independent experiments, n = 3). c Continuous treatment with 1 µM S1P enhanced migration speed of SGPL1-deficient RH-30 cells (black, gray and red line), whereas SGPL1 overexpression significantly inhibited the migration capacity during continuous S1P stimulation (green line). Mean ± SD, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, significantly different compared to mock transfected cells, unpaired t-test. d Native SGPL1 overexpression inhibited the ability for adherent colony formation in the presence of S1P, significantly (green bar). Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, significantly different compared to mock transfected cells, unpaired t-test. e Immunofluorescence-based analysis of the different localisation and distribution pattern of the mutated SGPL1 (left image: red fluorescence; right image: green fluorescence) and native SGPL1 (restored SGPL1: green GFP signal) in RH-30 cells. Note, the SGPL1 variants are not co-localized. Native SGPL1 overexpression restored the ER-association of SGPL1, whereas the mutated SGPL1 showed a diffuse cytoplasmatic localization