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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels

Fig. 6

HuD and GRB-10 increase markers of autophagy in neuroblastoma cells. A Determination of physical binding between mTOR and Raptor (mTORC1 component) by immunoprecipitation followed by Western blotting with representative antibodies. Full-length blots are presented in Supplementary Fig. S10. B Western blot analysis for mTORC1 activity and autophagy markers (control vs. silenced HuD and/or overexpressed GRB-10 in absence or presence of rapamycin). Full-length blots are presented in Supplementary Fig. S10. C Validation of autophagy induction by Western blot and relative quantifications shown (control vs. silenced HuD and/or overexpressed GRB-10). D Viability assay for control or silenced HuD (HuD siRNA) and/or overexpressed GRB-10 (GRB-10 OX) and/or rapamycin in IMR-32 cells. E Validation of autophagolysosome formation with marker MDC for control vs. silenced HuD (HuD siRNA) and/or GRB-10 (GRB-10 OX) and/or rapamycin; relative quantifications shown. F Autophagolysosome formation with MDC staining (in green) (control vs. silenced HuD and/or rapamycin); scale bar corresponds to 50 μm. Relative quantifications are shown (right). G Western blot analysis for mTORC1 activity and autophagy markers (control or overexpressed HuD) in HuD low expresser SK-N-MC cells and transfection of pCAGIG-HuD in SK-N-MC cells at different time point. Full-length blots are presented in Supplementary Fig. S10. GFP expression was observed under fluorescent inverted microscope; scale bar corresponds to 50 μm. Data are presented as mean ± SEM; t-test: *p < 0.05, **p < 0.01, ***p < 0.001

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