Antibodies and Reagents:
Antibodies against p-RelA-S536 (#3033; 1:1000), RelA (#8242; 1:1000), RelB (#4922, 1:2000; IHC: #10544), c-Rel (#4727; 1:1000), NF-ΚB2 (#3017; 1:1000), LKB1 (#3050; 1:1000), and NUAK1 (#4458; 1:1000) were purchased from Cell Signalling Technology (Danvers, MA). Antibodies against lamin A/C (MAB3211; 1:500) and actin (A2066; 1:20000) was purchased from Millipore (Temecula, CA, USA). Antibody against tubulin (T5168; 1:20000) was purchased from Sigma. HRP-conjugated antibodies against mouse IgG (NA931; 1:10000) and rabbit IgG (NA934; 1:10000) were purchased from Cytiva. All antibodies were diluted in tris-buffered saline-Tween 20 containing 5% bovine serum albumin, with the exception of NUAK1 (tris-buffered saline-Tween 20 containing 5% nonfat milk). BAY 11-7082 was purchased from Cayman Chemical (cat. 10010266; Ann Arbor, MI, USA). ROS-Glo H2O2 Assay was purchased from Promega (cat. G8820).
Generation of STK11KO and NUAK1KO cell lines:
Generation of OVCAR8-STK11KO16 and OVCAR8-NUAK1KO20 cell lines has been previously described. For FT190-NUAK1KO cells, two independent 20-nucleotide guide sequences targeting the NUAK1 gene 5’-GTGGC GGGGG ACCGC CCCGA-3’ (site 1) and 5’-GGGTC TCCTG CAGCT CGTAG CGG-3’ (site 2) were selected using the CRISPR Design Tool (http://tools.genome-engineering.org). Complementary oligonucleotides 5’-CACCG TCGGG GCGGT CCCCC GCCAC-3’ and 5’-CACCG GGGTC TCCTG CAGCT CGTAG-3’ for site 1 and 5’-CACCG GGGTC TCCTG CAGCT CGTAG-3’ and 5’-AAACC TACGA GCTGC AGGAG ACCCC-3’ for site 2 (Sigma-Genosys) were annealed and ligated into the BbsI-digested restriction endonuclease site of pSpCas9(BB)-2A-Puro plasmid76 (gift from Dr. F. Dick, Western University) to generate the pSpCas9-sgNUAK1-1 and -2 plasmids. Cells were seeded at 200,000 cells/well into 6-well plates and transfected with 0.5 µg each of pSpCas9-sgNUAK1 plasmids using LipofectAMINE2000 (Invitrogen) according to the manufacturer’s instructions. Media containing 1 µg/mL puromycin was replaced the following day, and cells were treated for one day. After growth recovery, the cells were trypsinized, counted, and seeded into 96-well plates to perform limiting dilution subcloning of NUAK1-knockout cells. Single colonies were expanded for protein isolation and the confirmation of NUAK1 loss by immunoblotting. Four clones lacking NUAK1 protein expression were identified by immunoblotting and pooled to generate a mixed population.
For experiments in Figures 1-3, 4A-D, 6, and Supplementary Figures S1A, S3, S4A-B, OVCAR8-NUAK1KO clone 62 was used. For experiments in Figures 4E-H and Supplementary Figures S1C, S4C, and S5, a mixed population containing equal proportions of 3 OVCAR8-NUAK1KO clones (50, 62, 63) were used to confirm that results were not clone-specific.
Cultured cell lines:
OVCAR8, OVCAR8-STK11KO, OVCAR8-NUAK1KO, HeyA8, and HeyA8-STK11KO cell lines were cultured in RPMI-1640 (Wisent). iOvCa147, iOvCa147-STK11KO, FT190, and FT190-NUAK1KO cell lines were cultured in DMEM/F12 (Life Technologies). For all cell lines, growth medium was supplemented with 10% fetal bovine serum. OVCAR8 and HeyA8 cells were obtained from the American Type Culture Collection; iOvCa147 cells were generated by our research group as previously described77. Adherent cells were maintained on tissue culture-treated polystyrene (Sarstedt, Newton, NC, USA). Spheroids were maintained in Ultra-Low Attachment (ULA) cluster plates (Corning, NY, USA). The immortalized human fallopian tube secretory epithelial cell line FT19059 was provided by R. Drapkin (University of Pennsylvania, Philadelphia, PA). All cell lines were authenticated by short tandem repeat analysis performed by The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada) and routinely tested for mycoplasma using the Universal Mycoplasma Detection Kit (30-1012K; ATCC).
Microarray Analysis:
Cells were seeded into 10 cm tissue culture-treated dishes (1 × 106 cells in 12 mL; 1 dish per sample) or 6-well ULA plates (5 × 105 cells in 5 mL; 3 wells per sample). Spheroids were harvested for RNA purification 24h post-seeding; adherent cells were harvested 48h post-seeding. RNA was collected as described in RNA collection and purification, and two 100 ng/µL dilutions in nuclease-free water were prepared for each sample: 3 µL for Bioanalyzer analysis to confirm acceptable RNA prep quality, and 10 µL for Clariom S microarray analysis. Dilutions were frozen on dry ice and shipped to The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada) for further processing. Data analysis and export was performed using the Transcriptome Analysis Console 4.0.1 software package (Thermo Fisher).
RNA collection and purification:
RNA was extracted using the RNEasy Spin Column kit (cat. 74104; Qiagen) according to the manufacturer’s protocol, with the optional DNaseI (cat. 79254; Qiagen) treatment. Adherent cells were collected by aspirating medium and scraping cells into 600 µL Buffer RLT and stored at -80ºC until processing. Spheroids were pelleted at 800 g, 4ºC, medium was aspirated, and cells were lysed in 350 µL Buffer RLT and stored at -80ºC until processing. RNA concentration, A260/280, and A280/230 were determined using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific).
Gene Set Enrichment Analysis:
Sample signals were exported using the Transcriptome Analysis Console 4.0.1 software package and formatted according to GSEA guidelines (https://software.broadinstitute.org/cancer/software/gsea/wiki/index.php/Data_formats) for Text file format for expression dataset. Phenotype labels were generated according to the Categorical class file format. The following GSEA (ver. 3.0) parameters were used: gene sets database, “h.all.v6.2.symbols.gmt”; permutation type, “gene_set”; number of permutations, 1000; collapse/remap to gene symbols, collapse; chip platform, “Clariom_S_Human.r1.chip”, enrichment statistic, weighted; metric for ranking genes, Signal2Noise; gene list sorting mode, real; gene list ordering mode, descending; maximum gene set size, 500; minimum gene set size, 15; collapsing mode for probe sets, max_probe; normalization mode, meandiv; randomization mode, no_balance; omit symbols with no match, true; median for class metrics, false; seed for permutation, timestamp. For identifying significantly enriched gene sets, p-value and false discovery rate cutoffs of 0.05 and 0.25, respectively, were used. Venn diagrams in Figure 1A-B and Supplementary Figure S2A were generated using the matplotlib-venn package (https://github.com/konstantint/matplotlib-venn).
Quantitative Reverse Transcription PCR:
cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher) according to the manufacturer’s protocol using 2000 ng RNA per reaction, yielding a final volume of 20 uL per reaction. Reactions were incubated in a MyCycler thermocycler (BioRad) using the following settings: 25ºC for 10 min, 37ºC for 120 min, 85ºC for 5 min, hold at 4ºC until sample retrieval. Each reaction was then diluted with an equal volume of nuclease-free water. qPCR was performed using the Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies) according to the manufacturer’s protocol scaled to a 10 µL reaction volume. Reactions were assembled in a 96-well plate, sealed with adhesive plastic film, and then centrifuged for 20 s to remove bubbles. Cycling was performed in a QuantStudio 3 RT-PCR System (Thermo Fisher) using built-in settings for SYBR Green Chemistry, Fast mode. Data analysis was performed using QuantStudio Design and Analysis Software 1.4.3. Fold-change values relative to controls were calculated using using the 2−ΔΔCT method78. Primer sequences (Supplementary Table S2) were obtained from https://www.origene.com and purchased from Invitrogen.
Preparation of whole-cell, cytoplasmic and nuclear lysates:
For assessment of total p-RelA (S536), day 3 whole-cell lysates were generated from adherent cells cultured at a density of 0.5-3 × 106 cells in 13 mL medium (10 cm dish), or spheroid cells cultured at a density of 1-3 × 106 cells in 6 mL medium (35 mm ULA well). For assessment of NF- κB transcription factor nuclear abundance, day 1 whole, cytoplasmic and nuclear lysates were generated from adherent cells cultured at a density of 3.5-4.5 × 106 cells in 13 mL medium (10 cm dish), or spheroid cells cultured at a density of 1.25-1.5 × 106 cells in 5 mL medium (35 mm ULA well). Seeding numbers were chosen to obtain acceptable protein yields for each cell line.
Whole-cell lysates: Adherent cells grown in tissue culture-treated plates or dishes were collected by aspirating medium, washing 2x with cold PBS, and scraping into modified RIPA buffer. Spheroids (at least 1.5 × 106 cells per sample) were collected by transferring the cell suspension into a conical tube on ice, pelleting by centrifugation in a swinging bucket rotor (800 × g at 4ºC for 4 min), aspirating medium, resuspending in at least 10 mL cold PBS, pelleting, resuspending again in cold PBS, pelleting, and aspiration of PBS. Cell pellets were then lysed in modified RIPA buffer, vortexed, subjected to one freeze-thaw cycle, and clarified by centrifugation (max × g at 4ºC for 20 min).
Cytoplasmic and nuclear lysates: Adherent cells were washed 2x with cold PBS and scraped into cold PBS using a cell lifter, transferred to a 50 mL conical tube and pelleted by centrifugation in a swinging bucket rotor (800 × g at 4ºC for 4 min). Spheroids (at least 3 x 106 cells per sample) were collected into a conical tube and pelleted by centrifugation in a swinging bucket rotor (800 × g at 4ºC for 4 min), washed 2x in cold PBS, and pelleted by centrifugation. Cell pellets were resuspended in at least 450 µL cold hypotonic lysis buffer and incubated on ice for 50 min, vortexing (max) for 10 s every 10 min. The suspension was then pelleted by centrifugation (max × g at 4ºC for 3 min). The supernatant (cytoplasmic lysate; 90% of hypotonic lysis buffer volume used) was transferred to another microcentrifuge tube and left on ice. The remaining supernatant in the original microcentrifuge tube was aspirated. 1 mL of cold wash buffer was added, vortexed for 20 s, pelleted by centrifugation (max × g at 4ºC for 1 min), and the wash buffer was aspirated. This wash step was repeated and the pellet was lysed in 100 µL modified RIPA buffer and vortexed to generate the nuclear lysate. Cytoplasmic and nuclear lysates were then subjected to 1 freeze-thaw cycle and clarified by centrifugation (max × g for 20 min at 4ºC). Hypotonic lysis buffer: 20 mM HEPES (pH 7.4), 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.3% Triton X-100, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 1x SIGMAFAST protease inhibitor cocktail (S8820; Sigma), 10 mM beta-glycerophosphate. Fraction Wash buffer: 10 mM HEPES (pH 7.4), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1x SIGMAFAST protease inhibitor cocktail. Modified RIPA buffer: 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 1x SIGMAFAST protease inhibitor cocktail (cat. S8820; Sigma), 10 mM beta-glycerophosphate. Total protein concentration was determined using the Bio-Rad Protein Assay according to manufacturer’s instructions (cat. 5000006; Bio-Rad).
Immunoblot Analysis:
Immunoblotting was performed using the Bio-Rad Mini-PROTEAN II Electrophoresis System (Bio-Rad) according to manufacturer’s instructions using gels cast in-house (30% acrylamide/bis solution 37.5:1, cat. #1610158; Bio-Rad). Densitometry was performed using the Image Lab 6.0.1 software package (Bio-Rad).
Treatment with BAY 11-7082:
Cells were seeded into 10 cm tissue culture-treated dishes at a density of 2.5 × 106 cells in a volume of 12 mL. The following day, cells were pre-treated with drug or vehicle: medium was removed by aspiration and replaced with 10 mL medium containing 5 µM BAY 11-7082 or an equivalent v/v DMSO as a vehicle control. The following day, cells were trypsinized, counted and seeded at the required density in medium containing BAY 11-7082 to a final concentration of 5 µM or an equivalent v/v DMSO. Seeding densities: 24-well ULA spheroids, 0.5-1 × 105 cells in 1 mL; 6-well ULA spheroids, 0.5-1.5 × 106 cells in 5 mL; 96-well ULA spheroids, 1 × 104 cells in 80 µL; 10 cm adherent, 1.5-2.5 × 106 cells in 10-12 mL. Cell numbers were chosen to achieve acceptable yields.
Trypan blue spheroid viability assay:
Cells were seeded into 24-well ULA cluster plates (0.5-1 × 105 cells per well in a volume of 1 mL). Spheroids were collected into microcentrifuge tubes on ice and pelleted by centrifugation at 500 × g for 3 min. Medium was aspirated and the pellet was washed by resuspension in 500 µL PBS (without drawing cells up into pipette tip), pelleted again as described above, resuspended in 50-250 µL trypsin/EDTA, and incubated at 37°C with gentle agitation every 10 min until aggregates were no longer visible (10-30 min). Trypsin was then inactivated by adding an equal volume of FBS. Trypan Blue dye was added (volume equal to trypsin/EDTA + FBS) prior to counting the sample and gently mixed by pipetting. Cell counting was performed using a TC20 Automated Cell Counter (Bio-Rad).
CyQUANT viability assay:
Spheroids were collected into microcentrifuge tubes on ice using a beveled P200 tip and pelleted at 500 × g for 5 min at 4ºC. Medium supernatant was aspirated and the pellet was stored at -80ºC. Relative viability was assessed by CyQUANT Cell Proliferation Assay (cat. C7026; Thermo Fisher) using 2x CyQUANT GR dye concentration to extend the assay linear detection range as described in the manufacturer’s instructions, blank-subtracted, and used to normalize ROS-Glo luminescence.
Measurement of ROS levels in EOC spheroids:
Cells were pre-treated with BAY 11-7082 or DMSO as described in Treatment with BAY 11-7082. 24h after treatment, cells were trypsinized, counted and seeded into 96-well round-bottom ULA cluster plates at a density of 10 000 cells/well in 80 µL, maintaining the same concentration of DMSO or BAY 11-7082 (5 µM). At d7, ROS levels were assessed using the ROS-Glo H2O2 Assay (Promega) according to the manufacturer’s instructions. 20 µL of 125 µM H2O2 substrate was added to each well and returned to the cell culture incubator for 5 h. 100 µL of complete ROS-Glo detection solution was then added to each well and incubated for 20 min at room temperature on a rocker. 175 µL from each well was then transferred to an opaque white 96-well plate, and luminescence was measured using a Synergy H4 plate reader (Biotek; Winooski, VT, USA). After blank subtraction, ROS-Glo luminescence was normalized to CyQUANT signal measured from identically-seeded and -treated replicate wells.
Xenograft tumour histology and immunohistochemistry:
Previously archived formalin-fixed, paraffin-embedded tumours20 were sectioned at a thickness of 5 µm. Hematoxylin/eosin staining was used to visualize tissue architecture, and adjacent sections were immunohistochemically stained to assess RelA and RelB (1:200 dilution), using hematoxylin as a counterstain. Sectioning and staining were performed by the Molecular Pathology Core Facility at Robarts Research Institute (London, Ontario, Canada). Images of stained tumour sections were captured using an Aperio ScanScope slide scanner (Leica).
Immunohistochemistry quantification and scoring:
IHC analysis was performed using the Fiji distribution of ImageJ79. Images of individual stained tumour nodules were exported using the ImageScope software package (Leica). Empty space around nodules was erased using the Adobe Photoshop software package. Necrotic tumour regions were identified using the “Trainable Weka Segmentation” plugin80, confirmed by visual inspection, and cleared. RelA and RelB staining was then evaluated using the “IHC Profiler” plugin81 for the total image, and for nuclear regions of the image (identified by thresholding of the hematoxylin channel).
Transient knockdown of RELA and RELB:
Cells were seeded in 6-well plates (1.5 × 105 cells/well) and transfected using DharmaFECT1 (1/500 final dilution) as per manufacturer's protocol (Dharmacon) using a final total siRNA concentration of 10 nM. RELA ON-TARGETplus SMARTpool (L-003533-00-0020), RELB ON-TARGETplus SMARTpool (L-004767-00), or ON-TARGETplus Non-targeting Pool (D-001810-10) were used. 24h after transfection, 3 mL of medium were added to each well, for a total volume of 5 mL. 48 hours later, cells were trypsinized, counted and seeded for experiments. Immunoblot analysis was performed on d0 adherent and d7 spheroid cell lysates to confirm RELA and RELB knockdown. RT-qPCR was performed in d1 spheroid cell and d2 adherent cell RNA to confirm decreased NF-κB target gene expression.
Microscopy:
Phase-contrast images were captured using a Leica DMI 4000B inverted microscope. Images of immunohistochemically-stained xenograft tumour sections were captured using an Aperio ScanScope slide scanner (Leica).
Statistical Analysis:
Statistical analyses were performed using GraphPad Prism 6.05 (GraphPad Software). Specific analysis details are described in figure legends.