Reagents and antibodies
Primary antibodies used for immunofluorescence are as follows: aPKC (C-20; Santa Cruz Biotechnology, Inc.) at 1:200, Ki67 (M7240; Dako; gift from F. Giancotti, Memorial Sloan-Kettering Cancer Center, New York, NY) at 1:200, E-cadherin (13–1900; Invitrogen) at 1:500, ezrin (E13420; BD) at 1:250, laminin B2 (clone A5; EMD Millipore) at 1:100, and ZO-1 (33–9100; Invitrogen) at 1:500. Secondary antibodies used for immunofluorescence are as follows: Alexa Fluor 488 and 568 (Invitrogen) at 1:200, Cy3 (Jackson ImmunoResearch Laboratories, Inc.) at 1:200, and Cy5 (Jackson ImmunoResearch Laboratories, Inc.) at 1:100. Primary antibodies used for Western blotting were AKT (9272; Cell Signaling Technology) at 1:1,000, β-actin (clone AC-74; Sigma-Aldrich) at 1:10,000, B-Raf (C-19; Santa Cruz Biotechnology, Inc.) at 1:1,000, cyclin A (H-432; Santa Cruz Biotechnology, Inc.) at 1:500, cyclin B (H-433; Santa Cruz Biotechnology, Inc.) at 1:500, cyclin D1 (K0062-3; MBL International) at 1:1,000, cyclin E (HE12; Santa Cruz Biotechnology, Inc.), ERK1/2 (M5670; Sigma-Aldrich) at 1:2,000, Flag (clone M2; Sigma-Aldrich) at 1:2,000, GFP (clone 3E1; Cancer Research UK) at 1:1,000, myc (clone 9E10; Cancer Research UK) at 1:1,000, p16 (C-20; Santa Cruz Biotechnology, Inc.; gift from A. Koff, Memorial Sloan-Kettering Cancer Center, New York, NY) at 1:250, p21 (C-19; Santa Cruz Biotechnology, Inc.) at 1:250, p27 (C-19; Santa Cruz Biotechnology, Inc.) used at 1:250, phospho-AKT (4060S; Cell Signaling Technology) at 1:1,000, phospho-ERK (M8159; Sigma-Aldrich) at 1:1,000, phospho-myc (Ser62; 33A12E10; Abcam) at 1:250, phospho-RB (9308; Cell Signaling Technology; gift from F. Giancotti) used at 1:1,000, and RB (C-15; Santa Cruz Biotechnology, Inc.) used at 1:500. Secondary polyclonal antibodies conjugated for HRP for Western blotting were obtained from Dako and used at 1:5,000. Other reagents used were as follows: phalloidin 647 (Invitrogen) at 1:100, DRAQ5 (Cell Signaling Technology) at 1:1,000, Hoechst 33342 (Sigma-Aldrich) at 1 µg/ml, U0126 (Promega), PIK-90 (Selleck Chemicals; gift from M. Overholtzer, Memorial Sloan-Kettering Cancer Center, New York, NY), and PD0332991 (Selleck Chemicals).
Plasmids and cloning
pQCXIP GFP was made by subcloning (Age1–BamH1) GFP from pEGFP-C1 into pQCXIP. K-Ras V12 was subcloned (BamH1) from pBABE K-Ras V12 (Addgene plasmid 9052; W. Hahn, Dana Farber Cancer Institute, Boston, MA) into pQCXIP GFP. B-Raf V600E was amplified by PCR from pBABE B-Raf V600E (Addgene plasmid 17544; Hao et al., 2007
) with the following primers that contain attB1 and attB2 Gateway recombination sites: forward, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGCGCTGAGCGGTGGCG-3′; and reverse, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGGACAGGAAACGCACCATATC-3′. PI3KCA H1047R was amplified by PCR from pBABE HA PI3KCA H1047R (Addgene plasmid 12524; Zhao et al., 2005
) with the following primers that contain attB1 and attB2 Gateway recombination sites: forward, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGCGCTGAGCGGTGGCG-3′; and reverse, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTTCAATGCATGCTGTTTAATTGTGT-3′. B-Raf V600E and PI3KCA H1047R were recombined into pQCFLAP, a derivative of pQCXIN with Gateway att sites, GFP, and Flag tags (obtained from P. Jallepalli, Memorial Sloan-Kettering Cancer Center, New York, NY). pWZL blast c-myc was obtained from Addgene (plasmid 10674; Boehm et al., 2005
). pBABE puro cyclin D1 was obtained from J. Brugge (Harvard Medical School, Boston, MA; Debnath et al., 2002
Caco-2 cells were cultured in DME with sodium pyruvate (Memorial Sloan-Kettering Cancer Center core facility) supplemented with 10% FCS and antibiotics at 37°C in 5% CO2
. Because there have been conflicting studies about the status of BRAF
, we sequenced the genomic BRAF
locus to confirm that our Caco-2 cells are BRAF
wild type (Oliveira et al., 2003
; Hao et al., 2007
). To culture mature Caco-2 3D structures, 4-well coverglass-bottom slides (Labtek) were coated with 10 µl of a mix of 80% Matrigel (growth factor reduced; BD) and 20% collagen I (Cultrex) and incubated at 37°C for 30 min to solidify. A single-cell suspension (104
cells) containing 2% Matrigel was added to each coated well, and cultures were incubated for 10 d before being fixed and stained. To culture two-cell structures, 5 × 103
Caco-2 cells were embedded in a final concentration of 40% Matrigel, 1 mg/ml collagen I, and 0.02 M Hepes in 8-well chamber slides (BD). This mixture was solidified at 37°C and overlaid with 400 µl media and then incubated for 2 d before being fixed and stained.
Plasmids encoding shRNA to deplete c-myc and a control shRNA vector (SHC002) were obtained from Sigma-Aldrich. The sequence of shmyc 1 was 5′-CCGGCAGTTGAAACACAAACTTGAACTCGAGTTCAAGTTTGTGTTTCAACTGTTTTTG-3′ (TRCN0000039640). The sequence of shmyc 2 was 5′-CCGGCCTGAGACAGATCAGCAACAACTCGAGTTGTTGCTGATCTGTCTCAGGTTTTTG-3′ (TRCN0000174055).
Virus production, infection, and selection
293FT cells were cultured in high glucose DME (Gibco/Invitrogen) supplemented with 10% FBS, antibiotics, and 2 mM l-glutamine (Invitrogen) at 37°C in 5% CO2. Cells were grown to 90% confluency in 10-cm plates and transfected with 3 µg VSVG and either 3 µg pCPG gag pol and 3 µg retroviral vector for retrovirus production or 3 µg pDeltaR8.9 and 3 µg shRNA DNA construct for lentivirus production. The transfection reagent used was Lipofectamine LTX with the Plus reagent (Invitrogen) and Opti-MEM (Invitrogen). The medium on the cells was changed to be serum and antibiotic free before the transfection mix was added. After 3 h, the media were removed, and Caco-2 media were added. Virus-containing media were centrifuged at 900 rpm for 3 min and passed through a 0.45-µm filter (Sarstedt). For control, K-Ras V12, B-Raf V600E, and c-myc retrovirus, purified viral supernatants were aliquoted and stored at −80°C. For PI3KCA H1047R retrovirus and shRNA lentivirus, the purified viral supernatant was centrifuged for 2 h at 19,000 rpm at 4°C. The resulting virus pellet was resuspended in PBS, aliquoted, and stored at −80°C.
In preparation for infection, 1.5 × 105 Caco-2 cells were plated per well of 6-well plates. Virus concentrated in PBS was diluted to 1.5 ml in serum- and antibiotic-free medium. All viral mixes were supplemented with 8 µg/ml polybrene (Sigma-Aldrich) before being added to the plates and centrifuged for 30 min at 2,250 rpm. The viral media were discarded and replaced by fresh media. Selection was started 2 d later with 3 µg/ml puromycin (Sigma-Aldrich), 0.7 mg/ml G418 (EMD Millipore), or 5 µg/ml blasticidin (Invitrogen).
To collect 3D cultures for Western blotting, a single-cell suspension containing 10% Matrigel was plated on ultralow attachment plates (Corning) and collected by resuspension in ice-cold PBS containing 5 mM EDTA and 1 mM sodium orthovanadate (Fournier et al., 2006
). This suspension was centrifuged at 900 rpm for 3 min and rinsed with ice-cold PBS containing 1 mM sodium orthovanadate. The resulting pellet was lysed in ice-cold buffer (1% NP-40, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 5 mM Na3
, and 10 mM NaF) containing protease inhibitors (Roche). Cell lysates were centrifuged at 18,000 g
for 5 min at 4°C, and the supernatant was immediately processed for SDS-PAGE and Western blotting. For lysis from 2D cell culture, cells were washed with ice-cold PBS containing 1 mM sodium orthovanadate and processed as described for 3D cultures.
Lysates were loaded on 4–12% Bis-Tris gels (Invitrogen) and run with MOPS buffer (Invitrogen). Gels were transferred onto nitrocellulose (Whatman) using transfer buffer (Boston BioProducts) containing 20% methanol (Thermo Fisher Scientific) for 1.25 h at 4°C on ice at 100 V. Membranes were blocked in TBS (Thermo Fisher Scientific) containing 0.1% Tween 20 (Sigma-Aldrich) and 5% milk. Primary antibodies were incubated overnight at 4°C in blocking buffer. Membranes were rinsed 3× for 15 min, incubated with HRP-conjugated secondary antibodies for 1 h, and rinsed 4× for 10 min before a 1-min incubation with ECL (GE Healthcare) and exposure onto film. Bands were quantified using ImageJ (National Institutes of Health).
Immunofluorescence and imaging
Before fixation, embedded 3D cultures were incubated with 50 U/ml collagenase I (Sigma-Aldrich) diluted in PBS for 15 min at RT. Both embedded and mature structures were fixed in 10% formalin (Sigma-Aldrich) for 30 min at RT. After triple washes with PBS, cells were incubated with immunofluorescence buffer (131 mM NaCl, 7 mM dibasic heptahydrate, 3 mM monobasic monohydrate, 8 mM sodium azide, 0.1% BSA, 0.2% Triton X-100, and 0.02% Tween 20) for 15 min at RT. Cells were next incubated in immunofluorescence buffer supplemented with 0.5% Triton X-100 and 1% BSA for 15 min at RT followed by triple washes of PBS. Primary antibody incubation was prepared in PBS with 1% BSA and left at RT for 2 h for embedded structures or overnight at 4°C for mature structures. Cells were then rinsed with PBS 3× for 10 min before secondary fluorescent antibody incubation in PBS with 1% BSA along with phalloidin and a DNA stain of either Hoechst or DRAQ5. Cells were again rinsed with PBS 3× for 10 min. Embedded structures were mounted on coverslips using fluorescent mounting medium (Dako). Mature structures were stored in PBS containing 0.2% azide. This protocol was modified from a previously published study (Jaffe et al., 2008
Fixed and stained 3D cultures were imaged on a spinning-disk confocal microscope (UltraVIEW ERS; PerkinElmer) with an EM charge-coupled device camera (iXon+ 897; Andor Technology). Mature structures were imaged using a 40× objective (Plan Neofluar, NA 1.3, oil; Carl Zeiss), whereas embedded structures were imaged using a 63× objective (Plan Apochromat, NA 1.4, oil; Carl Zeiss). All imaging was performed with oil immersion at RT. Images were acquired and analyzed with MetaMorph (Molecular Devices). MetaMorph was also used to adjust the brightness and contrast of images as well as to make videos from confocal z stacks.
Cells were cultured as described for 3D lysates. Total cellular RNA was isolated from pellets of day 1 3D structures with a purification kit (RNeasy Mini; QIAGEN). RT-PCR was performed on 200 ng of total RNA using a RT-PCR kit (SuperScript One-Step; Invitrogen) for each sample. Quantitative PCR was performed using TaqMan reagents on the real-time PCR system (7500; Applied Biosystems). The Taqman probe Hs00905030_m1 was used to amplify c-myc, and Hs99999905_m1 was used to amplify glyceraldehyde 3-phosphate dehydrogenase.
Statistical significance was evaluated using Prism software (GraphPad Software). The unpaired t test was performed with two-tailed p-values and 95% confidence intervals.
Online supplemental material
Fig. S1 shows Western blot analysis of K-Ras V12, B-Raf V600E, and PI3KCA H1047R cell lysates and effects of U0126 or PIK-90 inhibitors. Fig. S2 shows c-myc up-regulation and RNAi depletion in K-Ras V12 and B-Raf V600E cells. Fig. S3 shows that promoting proliferation in control cells or inhibiting proliferation in K-Ras V12 cells does not affect 3D morphology. Videos 1 and 2 are stack videos depicting control or K-Ras V12 day 10 3D structures, respectively, stained with actin and DNA. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201202108/DC1