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. 2012 Jul 19;3(7):e351. doi: 10.1038/cddis.2012.92

Figure 5.

Figure 5

Gx15-070 induces autophagy and necroptosis in TET cells. (a) Inhibitors of pan-caspase (50 μM ZVAD-FMK), necroptosis (30 μM NEC1), autophagosome formation (5 μM 3MA) and autophagosome maturation (25 μM CQ) rescued Gx15-070-induced growth inhibition. Percentage of reduction in cell growth induced by 100 nM Gx15-070 was calculated from the MTS data in the presence or absence of the indicated inhibitors for the three TET cell lines and for the small cell lung cancer cell line H146 as control. (*one-way ANOVA post hoc P<0.05; experiments were repeated three times). (b) Time-course electron microscopic morphology of T1682 cells treated with Gx15-070 at the indicated concentrations. After 1-h treatment, lamellar bodies (yellow arrow) consistent with early signs of autophagy and mitochondrial structural changes with loss of cristae (orange arrow) were evident. After 6-h treatment, early signs of necroptosis with intact nuclear envelope (top panel), and mitochondrial swelling with loss of mitochondrial matrix and cytoplasm vacuolization were observed (bottom panel). After 48-h treatment, late stages of necroptotic cell death (yellow arrow) and advanced stages of autophagy (orange arrow) were evident. The bottom panel depicts advanced stage of autophagic cell death with cytoplasmic vacuolization and lamellar bound structures consistent with autophagosomes. (c) Gx15-070 (1 μM) induced LC3BII accumulation after 48-h treatments in TET cells (top blot). LC3BII accumulation was observed in T1682 cells 1-h after Gx15-070 treatment at a concentration as low as 100 nM (bottom blot). (d) BECN1 and RIPK1 knockdown rescues Gx15-070-induced growth inhibition. Western blot shows shRNA knockdown of Beclin1 (sh-BECN1-1 and sh-BECN1-2) and RIPK1 (sh-RIPK1-1 and sh-RIPK1-2) in T1682 and TY82 cell lines. Sh-FF2: shRNA control vector carrying anti-exogenous luciferase shRNA FF2 (top). MTS assay of BECN1 and RIPK1 knockdown cells treated with 250 nM Gx15-070 (bottom). *denotes statistically significant differences compared with control (P<0.05); experiments were repeated three times