Supplemental Figures 1-6 from JNK1 Inhibition Attenuates Hypoxia-Induced Autophagy and Sensitizes to Chemotherapy
Authors: Vasilevskaya Irina A. , Selvakumaran Muthu , Roberts David , O'Dwyer Peter J. 
Published: April 2023

<p>Figure S1. Oligonucleotide pairs containing portions of human JNK1 and JNK2 were used to create retroviral vectors for delivery of shRNAs into HT29 cells. Target sequences (underlined) were taken from Hui et al ( J Clin Invest, 2008,118:3943-53). Oligonucleotide pairs were synthesized, annealed at equimolar concentration, diluted and ligated into pSUPER-RetroPuro-DR, a modified form of pSUPER-RetroPuro (OligoEngine, Seattle, WA). The resulting vectors were transfected into Phoenix-Ampho packaging cell line, kindly provided by Dr. Anil Rustgi (University of Pennsylvania, Philadelphia, PA) using Fugene 6 reagent (Roche). Viruses were collected, purified, aliquoted and stored at -80oC. Figure S2. Functional JNK1 in necessary for induction of autophagy in HT29 cells. Cells were subjected to hypoxia and oxaliplatin (5 mM) for 24 hours. Acridine orange (AO) staining was performed as follows: cells were washed twice with PBS, stained with 1 μm/ml acridine orange for 15 min at 37 {degree sign}C, washed again thrice and observed under microscope in PBS solution. Custom light cube for AO staining was used with blue (ca. 450 nm) excitation and red (ca. 650 nm) emission filters. Under AO staining, the cytoplasm and nucleus fluoresce green, whereas the acidic compartments fluoresce bright red or orange-red Our results show lesser content and size of acidic vesicles (including autolysosomes) in HTJ1.3 cells, as compared to control and HTJ2.2 lines. Scale bar - 100 mm. Figure S3. Results of MTT assays in HT29-derived cell lines after retroviral introduction of shRNA for JNK1 (sJ1) or JNK2 (sJ2). Shown are the average values of IC50 derived from three independent experiments in triplicate. Bars represent standard deviation; ***, P <0.001. Figure S4. Apoptosis is maximally enhanced by hypoxia and CQ in the HTJ1.3 cell line. A, Cells were seeded on glass slides and subjected to hypoxia for 24 hours with or without chloroquine (3 mM), followed by staining using Apoptosis and Necrosis Quantification Kit (Biotium). Apoptotic cells were labeled with FITS-Annexin V (green), necrotic - with Ethidium Homodimer III (red). The whole width of each image corresponds to 100 mm. B, Apoptosis was quantified by manual count of stained cells in several fields (n=5). Graph represents the percentage of cells stained with Annexin. EtBr-stained nuclei showed lower count and almost always coincided with Annexin stain (suggesting late apoptosis), and only showed slighter higher count (1-2% more cells with only EtBr staining) where marked by a dot. *, P<0.05. Figure S5. Following hypoxia/reoxygenation treatment (24+24 hours, 5 mM of oxaliplatin), autophagy still inhibited in JNK1-deficient cell line, but apoptosis is activated equivalently in all cell lines, most likely due to persistent stress, exacerbated by ROS formation, after oxygen supply is restored. Figure S6. Acridine orange staining of Bcl-XL-deficient HT29 cells shows slightly lower accumulation of acidic vesicles, when compared to parental cell line, earlier during hypoxia. By 24 hours of hypoxic exposure, however, this difference is no longer detectable. Scale bar - 100 mm. Table S1. Cytotoxic interactions between autophagy inhibitor (CQ) and JNK inhibitor (CC-401) in colon cancer cells.</p>