MOESM2 of Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

Additional file 2: Figure S2. (A) RT-PCR showing XBP1-u and XBP1-s expression levels in DMSO, tunicamycin, sunitinib and sunitinib+chloroquine treated Panc3.27 cells. Sunitinib or sunitinib+chloroquine treatment does not alter the expression of XBP1-s. (B) Histological analysis of mice treated with chemotherapeutics, sunitinib and chloroquine. The dual combination of Chemo+chloroquine, Chemo+sunitinib and the triple treatment of Chemo+sunitinib+gemcitabine shows noticeable reduction of ductal carcinoma (arrow). Scale bar: 10 ¾m. Sham vehicle control, Chemo gemcitabine plus paclitaxel, Sun sunitinib, CQ chloroquine

Untargeted mass spectrometry discloses plasma solute levels poorly controlled by hemodialysis

<div><p>Many solutes have been reported to remain at higher plasma levels relative to normal than the standard index solute urea in hemodialysis patients. Untargeted mass spectrometry was employed to compare solute levels in plasma and plasma ultrafiltrate of hemodialysis patients and normal subjects. Quantitative assays were employed to check the accuracy of untargeted results for selected solutes and additional measurements were made in dialysate and urine to estimate solute clearances and production. Comparison of peak areas indicated that many solutes accumulated to high levels in hemodialysis patients, with average peak areas in plasma ultrafiltrate of dialysis patients being more than 100 times greater than those in normals for 123 features. Most of these mass spectrometric features were identified only by their mass values. Untargeted analysis correctly ranked the accumulation of 5 solutes which were quantitatively assayed but tended to overestimate its extent. Mathematical modeling showed that the elevation of plasma levels for these solutes could be accounted for by a low dialytic to native kidney clearance ratio and a high dialytic clearance relative to the volume of the accessible compartment. Numerous solutes accumulate to high levels in hemodialysis patients because dialysis does not replicate the clearance provided by the native kidney. Many of these solutes remain to be chemically identified and their pathogenic potential elucidated.</p></div

MOESM4 of Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

Additional file 4: Figure S4. Survival analysis of Sunitinib, Chloroquine and Gemcitabine combinatorial treatment on orthotopic Panc02 murine models in vivo. (A) Cumulative survival (B) Mean overall survival. Mice exhibiting clincial PDAC growth were treated with various combinations of sunitinib (Sun), chloroquine (CQ) and gemcitabine/paclitaxel (Chemo). The triplet combination shows highest survival rate (p < 0.001). The Panc02 orthotopic model showed significantly increased mean survival for either of the single treatments of Chemo, Sun or CQ, compared to the sham groups (p < 0.05). Overall, the Panc02 model showed greater sensitivity to the combination drugs and longer survival compared to Kpcp1 models with the triplet combination resulting in longer than 4 months survival. Sham vehicle control, Chemo gemcitabine plus paclitaxel, Sun sunitinib, CQ Chloroquine. *: p < 0.05, **: p < 0.01

Kinetic behavior of chemically identified solutes.

<p>Kinetic behavior of chemically identified solutes.</p

Results of quantitative compared to untargeted mass spectrometric analysis.

<p>Results of quantitative compared to untargeted mass spectrometric analysis.</p

MOESM1 of Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

Additional file 1: Figure S1. Diagrammatic representation of ER stress and autophagy pathway and the drug targets that are used in this study to intervene the ER-autophagy pathway

MOESM3 of Inhibition of endoplasmic-reticulum-stress-mediated autophagy enhances the effectiveness of chemotherapeutics on pancreatic cancer

Additional file 3: Figure S3. IHC staining with anti-active Casp3 showing differentially increased apoptosis in the pancreas of drug treated orthotopic murine model: The murine PDAC tissue from the control group shows the lowest proportion of active Casp3 positive cells (arrow) in the pancreatic tissue. Both the Sun+CQ, and Chemo+CQ doublet treatment and the Chemo+Sun+CQ triplet treatments robustly increases the apoptotic cells in the ductal carcinoma region (p = 0.037, 0.004, 0.0006, respectively). As shown in the bar-chart, neither sunitinib alone, nor chemo alone could alter apoptosis significantly (p = 0.089, 0.12 and 0.071, respectively). However, there was a statistically significant increase of active Casp3 positive cells in the triplet treatment group when compared to all of the double treatment groups of Chemo+Sun (p = 0.0007), Chemo+CQ (p = 0.008) and Sun+CQ (p = 0.019). Sham vehicle control, Chemo gemcitabine plus paclitaxel, Sun sunitinib, CQ Chloroquine. *: p < 0.05, **: p < 0.01. Scale bar: 10 µm

Ligand-induced changes in the vestibule and grotto region of luciferase.

<p>The “active” DSLA bound (left, 2D1S.pdb) and ATP bound (right, 2D1Q.pdb) structures are compared from the same viewing points. Upper panels show a cross section through the vestibule and grotto region beyond the luciferin binding pocket (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g007" target="_blank">Fig. 7</a>). The lower panels show the vestibule as viewed from the luciferin pocket; the left panel shows the O10 of DLSA for orientation. Water molecules are shown in red in the +ATP structure and in gold in the DSLA structure (one of the eight water molecules is partially hidden in panel C and is indicated with a circle). The cross-section surface capping is semitransparent. Surface coloring is grey for carbon, red for oxygen and blue for nitrogen. DSLA (gold carbons in A) is a surrogate for luciferin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g001" target="_blank">Fig. 1B</a>). Similar changes are seen in American luciferase (3IES.pdb and 3IEP.pdb).</p

Identification of photolabeled residues in the peptide Gln-340 – Lys-366.

<p>Conditions are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g004" target="_blank">Fig. 4</a>. <b>A.</b> 3-azibutanol (1 mM) photolabels Asp-358. <b>B.</b> TFD-benzyl alcohol (100 µM) photolabels Ser-349.</p

The structure of Japanese luciferase in the vicinity of the main photolabeled residues.

<p>The structure is shown as a cross section through the luciferin pocket with DSLA bound in the luciferin and ATP sites (2D1S.pdb). Panel A shows a close up view. Panel B is the same view zoomed out (note the 10 Å scale bars) to show the luciferin and ATP pockets. Panel C was obtained by rotating panel B 180° on the y-axis without scaling, viewing the pocket from the opposite side. Cross sections through the protein are capped in white mesh, revealing the residues behind. DSLA is shown with golden carbons. The main photolabeled residues are shown in ball & stick representation with cyan carbons; note that Ser-316 shows two rotamers with each oxygen assigned half occupancy. Important residues for the activity of luciferase that may have been photolabeled are shown with green carbons in stick representation. Hydrogen bonds to ligands and water are shown in panel A by green dashed lines. Surface colors are: white, carbon (except for photolabeled residues, cyan or green); blue, nitrogen; red, oxygen. The upper inset in panel A shows the interaction between Ser-349 and DSLA and the hydrogen bonded water. The lower inset shows the hydrogen bonding network linking the major residues interacting with DSLA through water molecules. Hydrogen bonds shown range from 2.5 to 3.0 Å in length.</p