Dulbecco modified eagle medium (dmem)

Human CRC cell lines HCT116 (CCL-247, ATCC, Manassas, VI, USA) and HT-29 (HTB-38, ATCC) were maintained in McCoy’s 5A supplemented with 5% FBS (Wisent, Saint-Bruno, QC, Canada). SW48 (CCL-231, ATCC) and Caco-2/15 cell lines (A. Quaroni, Cornell University, Ithaca, NY, USA) were maintained in DMEM (Wisent) containing 10% FBS. IEC6 BRAF^V600E:ER cells [12 (link)] were cultured in DMEM without phenol red supplemented with 5% charcoal-stripped FBS (Wisent).

Bone marrow-derived macrophages were generated as previously described [41 (link)]. Briefly, mice were euthanized, tibias and femurs isolated, and the ends of each bone cut off. The tibia and femur from each leg were placed into a sterile 0.5 mL microfuge tube that had a hole punctured in the end with an 18-gauge needle, which was then placed inside of a 1.5 mL microfuge tube before the addition of 100 µl of DMEM (Wisent) to the 0.5 mL tube. Bone marrow cells were collected by centrifuging at 4,000 rpm for 5 min, resuspended, filtered, and plated in 80 mL of DMEM supplemented with 10% FBS (Wisent) and 1% penicillin/streptomycin (Thermo Fisher) in a T175 flask, and incubated at 37 °C in a humidified atmosphere at 5% CO₂. After 4 h, cells were plated in 15 cm tissue culture dishes in the presence of 20% L929 medium (as a source of macrophage colony stimulating factor) and left to differentiate for 7–8 days. One day prior to the experiment, cells were lifted into suspension in the existing L929-supplemented DMEM by gently scraping and seeded into the appropriate plate for subsequent experiments.

Immortalized neonatal rat astrocytes were established in house by SV-40 transfection of primary neonatal rat astrocytes (SV-NRA), isolated from 2 to 4 day old Sprague–Dawley rats. The SV-NRA were grown to 80% confluency in DMEM (Wisent, St-Bruno, QC, Canada) containing 10%FBS (Fisher Scientific, Hampton, NH) and Antibiotic/Antimycotic (Wisent, St-Bruno, QC, Canada). The cells were washed twice with HBSS (Wisent, St-Bruno, QC, Canada) and incubated in 10 ml DMEM and 1% FBS per T75 flask for 72 h. The astrocyte conditioned medium (ACM) was collected, pooled, filter sterilized and aliquoted for storage at −20 ℃.

All cell lines used in this study were acquired from ATCC, apart from HIEC and Caco-2/15 which were from N. Rivard. CRC cells were grown in Dulbecco’s Modified Eagle Medium (DMEM)(Wisent, St-Bruno, Qc, Canada) or in 50% DMEM/50% Ham’s F12 media (Wisent) for LoVo and T84 cells. Both media were supplemented with 10% Fetal Bovine Serum (Wisent) and cells grown at 37 °C in 5% CO₂. Plasmid transfection was performed using JetPRIME (Polyplus transfection, Illkirch, France), following the manufacturer’s instructions. siRNAs were transfected at a final concentration of 20 nM, except in HT29 (10 nM), using Dharmafect 1 and by following the manufacturer’s instructions. siRNAs were purchased from Dharmacon: RAB21(1) (J-009450-05), RAB21(2) (J-009450-08), ATG5 (J-004374-08) and VAMP8 (J-013503-05). Bafilomycin A1 was used at 200 nM in all experiments and cells were incubated either in normal growth media, in full starvation media (EBSS) or in glucose starvation media (DMEM without glucose, supplemented with 10% FBS) for the indicated time.

Vero cells (ATCC) and HT1080 cells (ATCC) were cultured in Minimum Essential Medium (MEM, Sigma,) and Human Embryonic Kidney HEK293T (ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Wisent). All culture media were supplemented with 10% Fetal Bovine Serum (FBS, Sigma), 0.3 mg/mL L-glutamine, 100 U/mL penicillin, and 100μg/mL streptomycin (Wisent). Cells were maintained at 37°C in 5% CO₂ at 100% relative humidity.
BMDMs were isolated from C57Bl/6J mice (stock no. 00064, originally purchased from Jackson Laboratories and maintained as a colony at the University of Ottawa) and differentiated as previously described [73 (link)]. Briefly, bone marrow cells were seeded in DMEM supplemented with 10% FBS (Wisent), penicillin, and streptomycin (Hyclone, GE healthcare). L929-conditioned media (20%) was utilized for macrophage differentiation, and Roswell Park MEMorial Institute (RPMI, Wisent) media supplemented with 10% FBS (Sigma-Aldrich), 0.3mg/mL L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Wisent) was utilized for seeding and subsequent experiments.

Murine macrophage-like cells (RAW 264.7: ATCC-TIB 71) were seeded at 10⁶ cells/well in 12-well plates in Dulbecco's Modified Eagle's medium (DMEM) (Wisent Bioproducts) supplemented with 10% fetal bovine serum (FBS: Wisent Bioproducts). Transformed YS1646 were diluted in DMEM-FBS to give a multiplicity of infection of 100 and centrifuged onto the monolayer (110xg for 10 min) to synchronize the infection. After 1 hour at 37°C in 5% CO2, plates were washed three times with phosphate buffered saline (PBS: Wisent Bioproducts) and replaced in the incubator with DMEM-FBS containing 50 μg/mL gentamicin (Sigma-Aldrich) to kill any extracellular bacteria and prevent re-infection. After 2 hours, the cells were washed with PBS three times and the gentamicin concentration was lowered to 5 μg/mL. After 24 hours, the cells were harvested, transferred to Eppendorf tubes and centrifuged (400xg for 5 min). Pellets were prepared for western blotting as above. For imaging experiments, RAW 264.7 cells were seeded into 6-well chamber slides at 10⁴ cells/well and cultured as above. After 24 hours, the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (Thermo Fisher), fixed with 4% paraformaldehyde in PBS and incubated for 10 min at RT. Images were obtained using a Zeiss LSM780 laser scanning confocal microscope and analyzed using ZEN software (Zeiss, Oberkochen, Germany).