Supplementary Materials and Methods Immunoblotting Immunoblot analysis was performed as described previously (1). Due to high-molecular weight of MUC4 (~ 950 kda) and MUC1 (~ 250 kda) proteins, electrophoresis was performed on 2% SDSagarose gel, whereas molecules less than 250 kda were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) under reducing conditions and blotted onto a PVDF membrane (Millipore). Membranes were incubated overnight at 4 0 C with primary antibodies (Table S1). Blots were washed and probed with peroxidase-conjugated secondary antibodies, and visualized using enhanced chemiluminescence (ECL) method (Thermoscientific). Quantitative RT- PCR RNA was isolated using QIAGEN RNeasy mini kit (Qiagen, Valenica, CA, U.S.A.) and cdna was synthesized using 2 μg RNA, random hexamer primers, and Super Script II RNase reverse transcriptase (Invitrogen) and real-time PCR was performed for MUC4 and MUC1. β-actin was used as an internal control. The primer sequences used: MUC4_forward: 5 - CGCGGTGGTGGAGGCGTTCTT-3 ; MUC4_reverse: 5 -GAAGAATCCTGACAGCCTTCA-3 ; β- actin_forward: 5 -ACCCCGTGCTGCTGACCGAG-3 and β-actin_reverse: 5 -TCCCGGCCAGC CAGGTCCA-3. The relative expression was calculated using 2 ΔΔCT method (2). Measurement of florescence to analyze ROS levels, autophagic vacuoles and cell death To analyze ROS production, PC cells were incubated with 1μM 2-7 -Dichlorofluorescein diacetate (DCFDA) (Sigma-Aldrich) for 15 min (3). After three washes with PBS, cells were collected in 500 μl of PBS and analyzed (at 488 nm) using flow cytometry. For the detection of autophagic vacuoles (positive for MDC) (Suppl. Fig 4A), cell lines were incubated with 50 μm of MDC at 37 C for 10 minutes. Following, cells were washed thrice with PBS and florescence was measured immediately at 300 nm using florescence reader (Biotek, SMATBLD). To quantify number of apoptotic and necrotic cells, PC cells were serum starved for 12h, followed by treatment of CD18/HPAF cells with 1% hypoxia, either alone or in
the presence of NAC (2.5 mm) or CQ (50 um). After the completion of treatment, Annexin-V-cy5 and propidium iodide (PI) (BD biosciences) staining were performed and analyzed by flow cytometry.
Reference List 1. Kumar S, Das S, Rachagani S, Kaur S, Joshi S, Johansson SL et al. NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer. Oncogene 2015; 34(37):4879-89. 2. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-408. 3. Torres MP, Rachagani S, Purohit V, Pandey P, Joshi S, Moore ED et al. Graviola: a novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism. Cancer Lett 2012;323(1):29-40.
Supplementary figures legends Supplemental Figure 1: Hypoxia reduces MUC4 protein expression independent of HIF-1α. (A) MUC4 expressing PC cell line, Colo357, was exposed to 1% hypoxia for 24 h. As anticipated, upon hypoxia treatment, HIF-1α expression was significantly induced, whereas MUC4 protein showed significant reduction, as compared to untreated controls. (B) The bar graph showing the percentage positive and negative expression for MUC4 and HIF-1α in stained PC tissue arrays. Supplemental Figure 2: Hypoxia-induced ROS reduces MUC4 expression by facilitating its degradation via autophagy pathway. (A) MUC4 expression was analyzed in lysates obtained from CD18/HPAF cell line treated with different concentrations of NAC for 24 h. (B) The bar graph showing mean florescence intensity (MFI) measured for DCFDA dye in indicated immortalized normal pancreatic and cancer cell lines. (C) Representative image showing increased autophagosome formation in H 2 O 2 and CoCl 2 (hypoxia mimetic) treated CAPAN1 cells. For the detection of autophagy vacuoles, MDC staining was performed. (D) To confirm an association between MUC4 and autophagy, autophagy was blocked in CAPAN1 cells (plated on the coverslips) by treating them with VB (10 µm) for 8 h. Cells were fixed and immunofluorescence staining was performed to look for the colocalization between MUC4 and LC3. VB treated PC cells exhibited increased expression and retention of MUC4 in accumulated LC3-positive vesicles. (Scale bar = 10 μm) Supplemental Figure 3: ROS reduces cell viability, which is partially rescued by MUC4 overexpression. (A) Immunoblots showing changes in the expression of p21 in hypoxia (1% O 2 ) treated T3M4 and CD18/HPAF cell lines. (B) Cell numbers were quantified after 24 h of H 2 O 2, NAC and NAC+H 2 O 2 treatment of CD18/HPAF and CAPAN1 PC cells. (C) CD18/HPAF and CAPAN1 cell lines were treated with different concentrations of exogenous H 2 O 2. Following treatment, MTT assay was performed to analyze the effect of treatment on cellular viability. (D) Immunoblot representing the ectopic expression of MiniMUC4 in MUC4 non-expressing MIA PaCa-2 cell line. (E) The
graphical representation to demonstrate the effect of 24 h and 48 h of combinatorial or individual treatment of H 2 O 2 and NAC on the proliferation of MIA PaCa-2/psectag and MIA PaCa-2/MiniMUC4 expressing cell lines. (*p<0.05 signifies statistically significant results; ns means insignificant changes; Scale bar =20 μm) Supplemental Figure 4: Increased autophagy in cancer cell lines is required for cellular viability. (A) The graph representing significantly high levels of autophagic vacuoles (measured by florescence measurement of MDC dye) in PC (CD18/HPAF and CAPAN1), colon (LS180) and head and neck (UMSCC1 and UMSCC10B) cancer cell lines compared to normal pancreatic ductal epithelial cells (HDPE). (B) MTT assay was performed to observe the effect of autophagy inhibitor, VB, on the viability of CD18/HPAF and CAPAN1 cells lines. (*p<0.05 means statistically significant; ns means no significant difference). Supplemental Figure 5: Clinical association between MUC4 and autophagy. (A) Histogram representation of the intensities plot for MUC4 and LAMP1. (B) Immunofluorescence analysis was performed in PC tissue section to observe the co-localization between MUC4 and LC3 molecules by confocal microscopy. (C) Histogram representing the intensities plots for MUC4 and 8-OHG molecules in stained PC tissue sections.