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Fig. 2 | Journal of Translational Medicine

Fig. 2

From: Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

Fig. 2

XBP1 splicing, lysosomal activities and cell viability in human PDAC cells treated with ER stress and autophagy modulating drugs. a Human PDAC derived Panc02.03 cells were treated with 2.5 µM tunicamycin and/or 25 µM STF for 72 h, and RT-PCR was performed on the extracted mRNA of these cells to monitor the XBP1 splicing and GRP78 expression. As expected, XBP1 splicing is increased in tunicamycin treated cells compared to DMSO treated cells, while co-treatment of STF-083010 reverses the upregulated splicing to the normal splicing levels, comparable to that of DMSO treated cells. GRP78 is increased with tunicamycin treatment, but is not further altered by the addition of STF-083010 treatment. b TEM images of DMSO (left) and tunicamycin-treated (middle) and STF-083010 (right) Panc02.03 cells, showing organelle pathology of ER vesicles (arrowhead), and lysosomes (arrow). Both tunicamycin and STF-083010 treated cells exhibit enlarged ER vesicles and have increased lysosomes in the cytoplasm. Bar-charts shows significantly increased lysosome contents in STF-083010 treated cells. Scale bar, 500 nM. c Panc02.03 cells were treated with tunicamycin (2.5 µM), STF-083010 (25 µM) with or without 4-PBA (100 µM) for 72 h, and the lysosomes were viewed by fluorescent microscopy using lysotracker staining. Both STF-083010 and tunicamycin treatments led to increased lysotracker staining, compared to DMSO treatment groups. Co-treatment with 4PBA reduced the elevated lysotracker staining to a comparable level with the DMSO treated group. Chloroquine at 50 µM causes cell clumping and cell death and reduced lysotracker staining. Scale bar: 5 µm. d Bar-charts showing normalized cell viability ratio of PDAC cells treated with STF-083010 (25 µM), chloroquine (50 µM), gemcitabine (250 nM) and various combinations (n = 4). Human PDAC-derived Panc02.03 cells were treated with various concentrations of single or combination treatments for 72 h and the viability was measured by Trypan Blue exclusion assay. STF-083010 and chloroquine show additive effects with gemcitabine (p < 0.01). Cell viability ratio was normalized with the viability of DMSO treated cells. *p < 0.05, **p < 0.01, CQ chloroquine, STF STF-083010, TM tunicamycin, Gem gemcitabine

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