Cells, transfections and reagents
Human HeLa and A549 cells were purchased from the American Type Culture Collection (ATCC) and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc., Waltham, MA USA). The stably transfected GFP-LC3B HeLa cell line was obtained as a gift from Yingyu Chen (Peking University, Beijing, China). Lentivirus-mediated shRNA knockdown of CMTM7 in A549 cells was done as described previously [5]. The cells were transfected with plasmids or small interfering RNAs (siRNAs) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA). Constitutively active Rab5 and dominant-negative Rab5 plasmids were kindly provided by Yingyu Chen (Peking University, Beijing, China). Based on the pcDNA.3.1/myc-His(-)B (Invitrogen, V85520) plasmid, pcDB-CMTM7 plasmid and pcDB-CMTM7-myc-His plasmid were constructed, and abbreviated them as CMTM7 and CMTM7-myc, respectively. Along with these, mCherry-ATG5, mCherry-WIPI-1 GFP-RHEBD60K and GFP-CMTM7 plasmids were also constructed in our laboratory. All plasmids were confirmed by DNA sequencing. siRNAs targeting Rab5 and VPS34 were designed and synthesized by GenePharma Co., Ltd. (Suzhou, China). The siRNA sequences were as follows: si-VPS34: 5′-GUGUGAUGAUAAGGAAUAUTT-3′, and negative control (si-NC): 5′-UUCUCCGAACGUGUCACGUTT-3′. Inhibition of autophagy was achieved by treating cells with 10 mM of 3-methyladenine (3-MA, Sigma-Aldrich, St. Louis, MO, USA) or 25 mM of chloroquine (CQ, Sigma-Aldrich, St. Louis, MO, USA). Other reagents used in this study were as follows: DAPI (Cell Signaling Technology, Inc., Beverly, MA, USA), Rapamycin (Selleckchem), and DMSO (Sigma-Aldrich, St. Louis, MO, USA).
Co-immunoprecipitation and Western blot analysis
Co-immunoprecipitation assays were performed with control and CMTM7-myc-overexpressing HeLa cells. Cells were harvested after transfection for 36 h in a buffer containing 20 mM Tris–HCl, at pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 1% protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were determined by using BCA protein assays (Pierce, Rockford, IL, USA). Whole-cell lysates (1000 μg) were incubated with the indicated antibodies for 12 h at 4 °C and further incubated for another 2 h after adding pre-equilibrated protein-G Sepharose beads (GE, New York City, NY, USA). The sepharose beads were then washed thrice with the same buffer at 4 °C. The beads were collected by centrifugation, and then resuspended in 2 × SDS loading buffer and heated to 100 °C for 5 min. The whole-cell lysates were then fractionated using 10% or 15% SDS–PAGE gels and then electrotransferred onto polyvinylidene difluoride membranes (GE, New York City, NY, USA). Western blotting was performed according to the standard protocol as previously described [5], and the following antibodies were used: anti-Rab5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Beclin1, anti-VPS34, anti-ULK1, anti-ATG14L, anti-UVRAG, anti-myc, anti-GFP, anti-ATG5, anti-RPS6KB1, anti-phospho-RPS6KB1Thr389, anti-ATG12, anti-4E-BP1, anti-phospho-4E-BP1Thr37/46, anti-AMPKα, anti-phospho-AMPKαThr172, anti-mTOR, anti-phospho-mTORSer2448, anti-ERK, anti-phospho-ERKThr202/Tyr204 (all from Cell Signaling Technology, Inc., Beverly, MA, USA), anti-LC3B, anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA), and anti-CMTM7 rabbit polyclonal antibody (Abcam, Cambridge, MA, USA). The protein bands were visualized by using a DyLight800-conjugated secondary antibody, and the signals were detected by an Odyssey Infrared Imager (LICOR Bioscience, Lincoln, NE, USA).
Confocal microscopy
Cells were incubated with 50 nM LysoTracker Red (Invitrogen, Grand Island, NY, USA) for 20 min at 37 °C, washed with PBS, fixed and permeabilized with 3% paraformaldehyde containing 0.1% Triton X-100 for 30 min at 4 °C. They were then washed with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI, 3 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA) for 10 min before being imaged with a TCS-SP laser-scanning confocal microscope (Leica Microsystems, Mannheim, Germany). The number of GFP-LC3B puncta per 20 cells was assessed, and statistical data were obtained from 3 independent experiments.
Xenograft nude mice model
All protocols regarding animals were reviewed and approved by the institutional Animal Research Ethics Board of Jilin University. Female BALB/c nude mice (6 weeks old, weighing 20 g) were maintained in a germ-free environment in the animal facility. The tumorigenesis assay was performed as previously described [40]. Briefly, CMTM7 knockdown or control A549 cells were trypsinized and suspended in PBS for subcutaneous injection. A total of 1 × 106 cells in 100 μL PBS were injected subcutaneously into the right axilla of nude mice (four mice per group). Tumor diameters were measured with a caliper every 3 days, and tumor volumes were calculated according to length × width2 × 0.5. The mice were sacrificed after 4 weeks, when their tumors were dissected, weighed and fixed for immunohistochemistry and transmission electron microscopy analysis.
Immunohistochemistry
Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tumor tissues as previously described [40]. Briefly, tissue sections were dewaxed, rehydrated and placed in 10 mmol/L citrate buffer (pH 6.0) before being heated in a microwave oven for 10 min twice. Sections were incubated with 3% H2O2 for 10 min, washed with PBS, blocked with 10% normal goat serum for 30 min, and then incubated with anti-Ki67 (1:800) (Cell Signaling Technology, Inc., Beverly, MA, USA) at 4 °C overnight. After being washed, the sections were stained with reagents of the Catalyzed Signal Amplification System Kit (Dako, Glostrup, Denmark). Photos were acquired with a Nikon E800 camera.
Transmission electron microscopy
A549 cells or tumor tissues were initially fixed in 0.1 M sodium phosphate buffer containing 3% glutaraldehyde (pH 7.4) at 4 °C and then fixed in 0.1 M sodium phosphate buffer containing 1% OsO4 (pH 7.2) for 2 h. The cells and tissues were dehydrated in an ascending series of acetone, embedded using an Ultracut microtome (LEICA ULTRACUT R, Bensheim, Germany) and sliced into 60 nm sections. Ultrathin sections were stained with uranyl acetate and lead citrate before being observed under a transmission electron microscope (TEM, H600IV electron microscope, HITACHI, Japan) at an accelerated voltage of 75 kV.
In vitro PI(3)P ELISA
Total amount of PI(3)P was examined in a quantitative and competitive ELISA format assay according to the manufacture (Echelon Biosciences K-3000). Briefly, VPS34 containing complex was enriched by anti-ATG14L antibody and then mixed with phosphatidyl inositol (PI) substrates in an appropriate reaction buffer. After the PI3K reactions were complete and quenched, reaction products were diluted and added to the PI(3)P-coated microplate for competitive binding to a PI(3)P detector protein. The amount of PI(3)P detector protein bound to the plate was determined through colorimetric detection.
Statistical analysis
The data are expressed as the mean ± s.d., and statistical analyses were performed using two-tailed Student's t-tests in Prism 5.0 (GraphPad Software, San Diego, CA, USA). Differences were considered significant if P < 0.05.