FIGURE 5.

SPHK1 inhibitor SKI-1a does not affect GNS cell proliferation or survival. A, expression of SPHK1, normalized to 18 S rRNA, was compared in a panel of GNS cell lines. The expression level is relative to the BAH1 cell line. B, the effect of SKI-1a and 1b on RN1 proliferation was assessed after 7 days by MTT assay. C, the effect of 500 nm SKI-1a or 1b on RN1 proliferation was assessed using the xCELLigence real-time growth assay system. D, the effect of SKI-1a on viable RN1 cell number after 3 days in the presence of temozolomide (TMZ), measured using an MTT assay. E, RN1 cells were cultured for 48 h in growth medium or HBSS in the presence of 500 nm SKI-1a, SKI-1b, or vehicle control (Veh). The proportion of non-viable cells was determined by flow cytometry. Proliferation and viability results are mean ± S.D. derived from a minimum of three treatments and are representative of two experiments. F, RN1 cells were pretreated for 24 h with 500 nm SKI-1a, SKI-1b, or vehicle control and then incubated for the indicated times in HBSS. Lysates were blotted with anti-LC3B, after which the membranes were stripped and reprobed with anti-α-tubulin as a loading control. The arrows indicate the lower LC3B-II band, a marker of autophagy. Some wells were treated for 24 h with 10 μm chloroquine (Chlq) as a positive control to block autophagosome turnover. V, vehicle control.