Antibodies and reagents
Anti-LC3B (1:2000) and anti-Tubulin (1:2000) (Sigma), anti-ATG5 (1:1000), anti-ATG7 (1:1000) and anti-phospho Ribosomal Protein S6 (Cell Signaling), anti- GAPDH (1:1000) (SCBT). The following inhibitors and reagents were used at the indicated concentrations, 3-methyladenine (3-MA) (Sigma) 3mM; Y27632 (EMD) 10µM and LysoTracker Red DND-99 (Invitrogen) 50nM.
pBabe-GFP-LC3, pBabe-mCherry-LC3, pMLP-IRES-shATG5 and 2XFYVEm-Cherry (FYVE domain from Hrs) were a kind gift from Dr. X. Jiang, MSKCC. Tandem pBabe-GFP-mCherry-LC3 was a kind gift from Dr. J. Debnath, UCSF. pRetro-LAMP1-GFP was cloned by inserting the rat LAMP1 sequence (from addgene plasmid#1817) into the BamHI-EcoRI sites of the pRetroQ-AcGFP1-N1 vector (Clontech). pBabe-CathepsinB-mCherry was cloned by inserting CathepsinB-mCherry into the BamHI-SalI sites of pBabepuro. The pBabepuro-Bcl2, and pLXSN-HPV-E7 constructs were a kind gift of Dr. J.S. Brugge, Harvard Medical School, and have been described elsewhere61
. The pBabe-H2B-mCherry and pBabe-mCherry-CAAX constructs were gifts of Dr. C. Leong and Dr. J.S. Brugge, Harvard Medical School.
Virus production and infection
For virus infection, cells were seeded in a 6 well plate at 5×104 per well. The next day 1ml viral supernatant was added with 10µg/ml polybrene for 24 hours followed by a media change. Cells were then selected with the appropriate antibiotic, puromycin (2µg/ml), G418 (200µg/ml), or blasticidin (10µg/ml).
siGenome SMART pool siRNAs and individual oligos against human VPS34, ATG5, ATG7 and FIP200 were obtained from Dharmacon, Chicago, IL. MCF10A cells were seeded in a 6 well plate at 6×104 per well and transfected with 100nM siRNA using Oligofectamine (Invitrogen). Cells were routinely assayed 48 hours post transfection.
MCF10A cells were cultured as previously described62
in DME/F12 + 5% horse serum, 20 ng/ml EGF, 10 µg/ml insulin or 100 ng/ml IGF-I, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 50 U/ml penicillin, and pen/strep. MCF10A cells overexpressing E7 and Bcl2 were generated as described61
. MCF7 cells were obtained from the Lombardi Cancer Center at Georgetown University, J774 mouse macrophages (ATCC) and HEK293 cells (ATCC) were cultured in DMEM + 10% FBS with pen/strep. U937 human monocytic cells were cultured in RPMI-1640 + 10% FBS with pen/strep.
To quantify entotic cell fate, 1.5×105
cells were seeded overnight on 35mm glass-bottomed dishes (MatTek, Ashland, MA) and cell-in-cell structures were imaged by time-lapse microscopy the next day. Fluorescence and differential interference contrast (DIC) images were acquired every 4 minutes for 20 hours using a Nikon Ti-E inverted microscope attached to a CoolSNAP CCD camera (Photometrics), and NIS Elements software (Nikon). Only live internalized cells at the start of time-lapse were quantified for cell fate. Internalized cells were scored for release, death or no change. Control experiments demonstrated that >95% of cell-in-cell structures chosen for analysis were completely enclosed and shielded from external media at the start of imaging, as assessed by FM4-64 staining. Entotic structures chosen by DIC were stained with FM4-64, which labels membranes in contact with external media. The numbers of structures staining positive or negative were quantified (Supplementary Fig. 3c
). The timing and type of internalized cell death was determined by DIC morphology, cessation of movement or, where present, monitoring nuclear markers. Inhibitors were added, as indicated, 30 minutes prior to image acquisition. For mixed cultures, H2B-mCherry expressing cells were plated with GFP-LC3 expressing cells at a 1:1 ratio. Confocal imaging was performed with the Ultraview Vox spinning disc confocal system (Perkin Elmer) equipped with a Yokogawa CSU-X1 spinning disc head, and EMCCD camera (Hamamatsu C9100-13), and coupled with a Nikon Ti-E microscope. All imaging with live cells was performed within incubation chambers at 37°C and 5% CO2
. Confocal image acquisition and analysis was performed with Volocity software (Perkin Elmer).
Cells were grown on grid etched Aclar film, and imaged by fluorescence microscopy until LC3 recruitment to vacuole membranes. Samples were then fixed in 2.5% Glutaraldehyde / 2% Paraformaldehyde in 0.075M Cacodylate buffer pH 7.5 for one hour followed by rinsing in Cacoldylate buffer and post fixation in 2% Osmium Tetroxide for one hour. The samples were then rinsed in double distilled water followed by dehydration in a graded series of alcohol 50%, 75% 95% through absolute alcohol and overnight in 1: 1 Proplene Oxide / Poly Bed 812. The samples were then embedded the following day, onto Poly Bed 812 filled Beem capsules and cured in an oven at 60°C for two days. Following immersion in liquid nitrogen, Aclar film was then ripped from the capsules and ultra thin sections were obtained with a Reichert Ulltracut S microtome. Sections were stained with Uranyl Acetate and Lead Citrate. Images were obtained using a JEOL 1200 EX Transmission Electron microscope.
Measuring autophagy using tandem GFP-mCherry-LC3
MCF10A cells expressing GFP-mCherry-LC3 were plated on glass-bottom coverslip dishes. Internalized entotic cells, or control single cells, +/− HBSS (12 hours), were imaged by confocal microscopy in the presence or absence of VPS34, ATG5 or FIP200 siRNA. Fluorescence intensities of mCherry and GFP were measured in cells using Volocity software. Data were normalized against background and mCherry/GFP ratios calculated.
Measuring autophagy by electron microscopy
MCF10A cells were seeded on non-adherent plates for 6 hours to induce a high frequency of cell-in-cell formation. Cells were then seeded onto glass coverslips overnight. Following this, cells were trypsinized, pelleted and fixed in 2.5% Glutaraldehyde / 2% Paraformaldehyde in 0.075M Cacodylate buffer pH 7.5 for one hour. Pellets were then processed as above. 3 or more fields of view were acquired within the cytoplasm of internalized or single cells, 10 individual cells were used in each group. The area of autophagosomes and autolysosomes was calculated as a percentage of cytoplasm using ImageJ software (NIH).
Apoptotic phagocytosis, latex bead, and macropinosome assays
For apoptotic phagocytosis assays, J774 cells were seeded onto glass-bottom coverslip dishes in the presence of 200U/ml IFNγ for 2 days. U937 cells expressing H2B-mCherry were stimulated with 80J/m2 to induce apoptosis and added to J774 cultures at 4×106 per dish. Where indicated, 100nM Concanamycin A was added before image acquisition. For latex beads (Polysciences, Inc, PA), 3µm beads were added to macrophage at a ratio of 10:1. Cells were monitored for 20 hours, and z-stacks were acquired every 10 minutes. For MCF10A macropinosome assays, 10kD red fluorescent dextran was added to culture media (0.1mg/ml) for 10min, followed by washing in media for 5min, and confocal imaging (0.5µm z-stacks) of red dextran-labeled macropinosomes at 1min intervals for 30min to monitor recruitment of GFP-LC3. For J774 macropinosome assays, cells were imaged by confocal microscopy in the presence of 10kD red dextran (0.1mg/ml). Lysotracker red (Invitrogen) was added to cultures (50nm) after the timelapse imaging to image acidification of phagosomes.
Imaging C.elegans apoptotic phagocytosis
The mCherry::RAB-5; GFP::LGG-1 expressing strain was generated by crossing the pie-1pr::mCherry::rab-5
allele from strain RT1043 into the lgg-1pr::lgg-1::gfp
expressing strain (DA2123). Both strains were provided by the Caenorhabditis
Genetics Center (University of Minnesota, Minneapolis, MN). RNAi experiments were performed by bacterial feeding as described previously63
. Embryos obtained from dissected gravid hermaphrodites were placed on 2% agarose pads and mounted on a coverslip for observation using an Ultraview Vox spinning disc confocal system (Perkin Elmer) equipped with a Yokogawa CSU-X1 spinning disc head, and EMCCD camera (Hamamatsu C9100-13), and coupled with a Nikon Ti-E microscope. For apoptotic corpse counting, embryos at different developmental stages, as assessed by morphology, were imaged by DIC. Corpses were identified by morphology and counted.
Cells were scraped into ice-cold RIPA buffer (50mM Tris pH 7.4, 150mM NaCl, 2mM EDTA, 1% NP40, 0.1% SDS + protease inhibitor cocktail) and lysed for 10 minutes on ice. Lysates were centrifuged 13,000rpm at 4°C for 12 minutes and protein quantified by BCA assay (Pierce, Rockford, IL). Samples were separated on 10% polyacrylamide SDS-PAGE gels (15% for LC3 blots), and transferred to a polyvinyldifluoride membrane. The membrane was blocked into TBS-T + 5% BSA and incubated overnight at 4°C with primary antibodies diluted blocking buffer. Blots were incubated with horseradish peroxidase conjugated to secondary antibodies and protein detected using enhanced chemiluminescence (Invitrogen). Densitometry analysis was carried out using ImageJ software (NIH).
Total RNA was prepared using the RNAeasy mini kit (Qiagen) 48 hours after transfection of cells with siRNA. Quantitative PCR was performed using the Bio-Rad iCycler real-time system (MyiQ), with SYBR green detection (iScript One-Step RT-PCR Kit with SYBR green (Bio-Rad)). Samples were analyzed by the standard curve method in triplicate. Reactions contained a single product as determined by agarose gel electrophoresis and melting curve analysis. The following primer pairs were used: ACTIN For AGAGCTACGAGCTGCCTGAC ; ACTIN Rev AGCACTGTGTTGGCGTACAG; FIP200 For GTGCTGGGACGGATACAAAT; FIP200 Rev TTTCCAATGCAAGCTGTGTC.
Cell-in-cell formation – suspension assay
MCF10A (1 × 105 cells) pretreated with Vps34 or ATG5 siRNA, or in the presence of 3-MA or Y27632 were plated in non-adherent 6-well dishes for 7 hours. Cytospins of cells were made and stained for E-cadherin and β-catenin. The percentage of cells in cell-in-cell structures was quantified by counting >300 cells per slide.
Colony formation in soft agar
5×104 MCF10A cells overexpressing E7 and Bcl2 were added to 1ml of growth medium with 0.3% agar and layered onto 1ml of 0.5% agar beds in six-well plates and covered in 1ml liquid growth media. Cells were fed with 1ml of liquid medium every 5 days for 2 weeks, after which colonies were stained with 0.02% iodonitrotetrazolium chloride (Sigma–Aldrich) and quantified using an Optronix Gelcount colony counter (Oxford Optronix Ltd., Oxford, UK). Assays were conducted in duplicate in three independent experiments. Where indicated, 3-MA (3mM) or Y27632 were included in all solutions. Counts of cell-in-cell structures were made by light microscopy, 48 hours after seeding in soft agar.
Indicated P values were obtained using the Student’s t-test, or Chi-square test where indicated.