Beta actin

Cells were lysed using mammalian protein extraction reagent (Thermo Scientific), supplemented with protease inhibitor and phosphatase inhibitor cocktail (Thermo Scientific). Protein content was determined with a Bradford assay after which 20 μg of protein sample was separated by sodium dodecyl sulfate (SDS)/PAGE, transferred to polyvinylidene fluoride (immobilon) membranes and blocked in 5% skimmed milk (Sigma) in Tris-buffered saline (TBS) containing 0.05% Tween20 (Sigma).
Immunodetection was performed with antibodies directed against BRCA2 (Calbiochem, #OP95), BRCA1 (Cell Signaling, #9010), RAD51 (GeneTex, #gtx70230), EMI1 (Invitrogen, #37-6600), γ-H2AX (Cell Signaling, #9718), PTIP (Abcam, ab70434) all diluted 1:1,000 and Beta-Actin (MP Biomedicals, #69100) diluted 1:10,000. Horseradish peroxidase-conjugated secondary antibodies (DAKO) were diluted 1:2,500 and used for visualization using chemiluminescence (Lumi-Light, Roche Diagnostics) on a Bio-Rad bioluminescence device, equipped with Quantity One/ChemiDoc XRS software (Bio-Rad). Uncropped versions of all western blots can be found in Supplementary Fig. 9.

Cultured cells were lysed in Mammalian Protein Extraction Reagent (MPER, Thermo Scientific), supplemented with protease inhibitor and phosphatase inhibitor cocktail (Thermo Scientific). Proteins were separated on SDS-polyacrylamide gels (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). Membranes were blocked in 5% milk or bovine serum albumin (BSA) in Tris-buffered saline, with 0.05% Tween-20. Immunodetection was done with antibodies directed against BRCA2 (1:1000, Calbiochem, #OP95), BRCA1 (1:1000, Cell Signaling, #9010), cGAS (1:1000, Cell Signaling, #15102), STING (1:1000, Cell Signaling, #13647), cMYC (1:200, Santa Cruz, sc40, Abcam (ab32072) 1:1000), pIRF3 (1:1000, Cell Signaling, # 29047), IRF3 (1:1000, Cell Signaling, # 4302), STAT1 (1:1000, Cell Signaling, # 9172), pSTAT1 (1:1000, Cell Signaling, # 8826) and beta-Actin (1:10.000, MP Biochemicals, #69100). Horseradish peroxidase-conjugated secondary antibodies (1:2500, DAKO) were used and visualized with chemiluminescence (Lumi-Light, Roche diagnostics) on a Bio-Rad Bioluminescence device equipped with Quantity One/Chemidoc XRS software (Bio-Rad).

The following primary antibodies were used for immunoblotting: AHR (Cell Signaling Technology, #13790, 1:1000), ASNS (Thermo Fisher Scientific, # PA5-56113, 1:1000), ATF4 (Cell Signaling Technology, #11815, 1:1000), beta-Actin (MP Biomedical, #691002, 1:1000), E-cadherin (Cell Signaling Technology, #3195, 1:1000), MMP24 (Genetex, #GTX128246, 1:1000), SMAD2 (Cell Signaling Technology, #5339, 1:1000), pSMAD2 (Cell Signaling Technology, #3108, 1:1000). Secondary antibodies were HRP-conjugated (Pierce Antibodies, 1:4000). For qRT-PCR, RNA was isolated and purified using High Pure RNA Isolation Kit (Roche, Mannheim, Germany), and reversely transcribed by Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Mannheim, Germany). ACTB, AHR, ASNS, ATF4, CYP1A1, GAPDH, HPRT1, MMP9, or MMP24 expression (primers listed in Supplementary Table 3) was quantified in duplicates or triplicates on a LightCycler®480 System (Roche, Rotkreuz, Switzerland) using the 2-ΔΔCt method and ACTB, GAPDH, and HPRT1 as internal controls.

V. cholerae (VC1) cells were grown with or without the presence of 100 μM tryptophol acetate at 30 ^oC for 16 h. Culture supernatants were obtained by centrifugation of these cultures at 1100xg for 10 min. The cell pellets lysed using × 5 Sample buffer (125 mM Tris, 0.25% BPB, 10% 2-Mercaptoethanol, 10% SDS, 50% Glycerol; GenScript, Piscataway, USA) for 5 min at 95 °C.
Protein concentrations for the analyzed cells extract samples were determined using Bio-Rad protein assay and then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer protocols (Bio-Rad). After incubation with 5% skim milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary goat anti-cholerae toxin sub unit B (1:500; 227040, Sigma-Aldrich) for 16 h at 4 °C. Membranes were washed three times for 10 min and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-goat secondary antibodies for 1 h at room temperature (A50-101P, Bethyl Laboratories, Montgomery, USA). Beta-actin used as loading control (1:1000, MP Biomedicals, Santa Ana, CA, USA). Blots were washed three times with TBST and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocols.

Day 1 adult worms were washed twice in PBS. Half the worm pellet was resuspended in RIPA buffers with protease inhibitors (Roche, Basel, Switzerland), lysed via sonication, and then used to determine protein concentration with a BCA assay (Pierce, Waltham, MA, USA). The other half of the sample was lysed via sonication in 2x Laemmli sample buffer (BioRad, Hercules, CA, USA) containing 5% beta-mercaptoethanol. From this lysate, 20 ug of each sample was loaded and separated with a 10% tris-glycine gel (BioRad). The separated proteins were transferred to a 0.2 μm nitrocellulose membrane (Invitrogen), then incubated in primary antibodies (phospho-Drosophila p70 S6 Kinase (Thr398), 1:500, Cell Signaling #9209, and beta-actin, 1:1000, MP Biomedicals #8691002) in TBS overnight. The membrane was incubated for 1 h in secondary antibodies (IRDye 800CW Goat anti-rabbit (LI-COR), 1:20,000 and IRDye 680RD Goat anti-mouse, 1:20,000 (LI-COR)). LiCor Odyssey CLx infrared imaging system was used to image the blot and the Odyssey Image Studio software (version 5.2, Lincoln, NE, USA) was used to quantify band intensity.

Adherent cells were exposed to hypoxic conditions for 24 hours in a hypoxic chamber (MACS VA500 microaerophilic workstation, Don Whitley Scientific, UK). The atmosphere in the chamber consisted of 0.2% O₂, 5% CO₂ and residual N₂. Normoxic dishes were incubated in parallel in ambient air with 5% CO₂. Protein isolates were prepared by scraping cells in RIPA, sonicating the samples, and centrifugation them to remove cellular debris. Protein concentrations were measured using Bradford protein quantification reagent (Bio-rad). Western Blot was performed using primary antibodies against CAIX (M75, kindly provided by Professor Silvia Pastorekova, Institute of Virology, Slovak Academy of Science, Slovak Republic), and beta-actin (MP Biomedicals, #691001) as a reference protein. Primary antibodies were incubated overnight at 4°C, whereas HRP (horseradish peroxidase)-linked secondary antibody (Cell Signalling, #7076) was incubated for 1 hr at room temperature (RT).