Figure 6. LDE225 increases autophagosome as well as autolysosome formation.

A. SP53 (Left) and Jeko (Right) cells were treated with LDE225 (30 μM) or DMSO in the presence or absence of lysosomal inhibitor CQ (20 μM) for 24 h. The difference in LC3-II levels in the presence or absence of CQ was larger under LDE225-treated conditions compared with cells under DMSO-treated conditions. Densitometric values of LC3-II were indicated in each lane after normalization to GAPDH using a Gel-Pro Analysis software. Increased values after CQ treatment indicate increased autophagic flux. B. Cells were treated with different dosages of LDE225 (0-50 μM) combined with autophagy inhibitor 3-MA (5 mM). The cytotoxicity at 72 h was determined by MTT assays. Data represent the mean ± S.D. from three independent experiments. **p < 0.01, ***p< 1E-05 (average viability of LDE225/3-MA-treated cells vs. average viability of DMSO-treated cells; Student's t-test). C. Fold changes in cell viability with or without 3-MA treatment are shown after normalization to DMSO-treated cells. Data represent the mean ± S.D. from three independent experiments. *p < 0.05, **p < 0.01, ***p< 1E-05 (vs. cells treated with LDE225 only; Student's t-test). D. MCL patient cells were treated with LDE225 (0-50 μM) or DMSO in the presence or absence of 3-MA (5 mM). The cytotoxicity at 48 h was determined by MTT assay. Data represent the mean ± S.D. from three independent experiments. **p < 0.01, ***p< 1E-05 (vs. cells treated with LDE225 only; Student's t-test). E. CXCR4 knockout (CXCR4^KO) cells were generated using the CRISPR-CAS9 system. LDE225 treatment failed to induce autophagy in CXCR4^KO cells (Top). Both Jeko parent cells and CXCR4^KO Jeko cells were treated under different dosages of LDE225 (5 μM-30 μM). The cytotoxicity at 48 h was determined using MTT assays. Data represent the mean ± S.D. from three independent experiments (Bottom). **p < 0.01 (vs. Jeko parent cells; Student's t-test).