Supplementary Figure 1.MRK003 Induced Apoptosis Gics

Tanaka et al. 1

Supplementary Figure Legends

Supplementary Figure 1.MRK003 induced apoptosis GICs.

(a) The representative flow cytometry data of AnnexinV-FITC apoptosis assay showed in relativelysensitive GICs such as 1123M, Me83, MD13 and TGS01. (b) AnnexinV-FITC apoptosis assay data for relativelyresistant GICs such as 157NS, 146NS and TGS04 was described.

Supplementary Figure 2.Myristoylated-Akt induction in relatively-sensitive GIC, 1123M was practically unable to rescue the cells from the effects of MRK003.

(a) Western blotting of phospho-Akt was demonstrated after MRK003 treatment for 48h. Although expression of phospho-Akt in the control-1123M was clearly decreased by MRK003, phospho-Akt expression in myrAkt-1123M was maintained. (b) Cell viability assay was performed after MRK003 treatment for 72h. A minor increase in the viability of myrAkt-1123M in comparisontothe control-1123M was observed in cells treated with 0.5μM MRK003 only. (c) Although sphere number did not change between myrAkt- and contro-1123M, sphere size of myrAkt-1123M was bigger than that of the control treated with 1μM MRK003. (d) No significantdifference in the results of the apoptosis assay was observed between myrAkt and control-1123M, as analyzed by AnnexinV-FITC. All experiments were conducted in triplicate, n=3. **P0.01 using 2-way ANOVA.

Supplementary Figure 3.CD44 and CD133 expression pattern of sensitive lines were different from that of resistant lines.

(a) Representative flow cytometry data of CD44-FITC was shown. (b) Representative data for flow cytometry of CD133-PE was shown.

Supplementary Figure 4.Other γ-secretase inhibitor, DAPT suppressed sphere forming ability of MRK0003 relative sensitive lines.

(a)Sphere number of 30R was significantly decreased a dose dependent fashion with DAPT treatment. Sphere number of TGS01 and TGS04 was not suppressed by DAPT treatment.(b) Sphere size of both 30R and TGS01was significantly decreased a dose dependent fashion with DAPT treatment. Sphere size of TGS04 was significantly decreased with DAPT 50 μM.Microphotographs were showntumorsphere treated DMSO (DAPT 0 μM) or DAPT.

*P < 0.05,**P0.01 using one-way ANOVA.Bar of microphotograph indicated 200μm.

Supplementary Figure 5.Summary of our analyses.

MRK003 suppressed cell viability and sphere formation ability, and induced apoptosis in patient-derived GICs. Sensitivity for MRK003 was divided into “relativelysensitive” and “relativelyresistant”. MRK003 treatment strongly decreased phospho-Akt expression level in relativelysensitive GICs, butphospho-Akt expression levelof relativelyresistant GICs was not suppressed by MRK003 treatment as same as relatively sensitive GICs. The effect of MRK003 partially depends on the inhibition of Akt. Since the relativelysensitive GICs exhibited high levels of CD44 and low levels of CD133, it may be suggested that MRK003 is effective in CD44high and CD133low GIC.