Cell culture and transfections
HeLa cells were grown and transfected as described previously 
. HCC-1395 (CRL-2324) and HCC-1954 (CRL-2338) cells were purchased from ATCC and grown in RPMI-1640 medium (GIBCO, Invitrogen) supplemented with 10% fetal bovine serum in a 5% CO2
atmosphere at 37°C.
Confocal fluorescence microscopy
Immunofluorescence microscopy was performed using HeLa, HCC-1395 and HCC-1954 as previously described 
. The following primary antibodies were used for immunofluorescence studies: rabbit anti-human FYVE-CENT antibody, used in 1
300 dilution, as described before 
, mouse anti-α-tubulin, used in 1
1000 dilution and purchased from SIGMA, rabbit anti-human Beclin 1 and mouse anti-human Aurora B antibody, both used in 1
200 dilution and purchased from Abcam. The secondary antibodies used were goat-anti-mouse Alexa Fluor® 488, in 1
500 dilution from Invitrogen and Cy3-labelled goat anti-rabbit antibody, in 1
500 dilution and Cy2-labelled goat anti-mouse antibody, in 1
200 dilution purchased from Jackson Immunoresearch. Alexa Fluor® 594 phalloidin, used in 1
750 dilution, and Hoechst 33342, used at 1 µg/µl, were purchased from Invitrogen.
To determine the cell-specific distribution of FYVE-CENT, Beclin 1, VPS34, beta-actin and the overexpressed TTC19 and KIF13A-myc tagged constructs, the various cell lines were lysed in lysis buffer (25 mM HEPES pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 5 mM EGTA, 1 mM DTT, 0.5% Nonidet P40, 1
100 proteinase inhibitor mix (Roche Applied Science). After centrifugation for 5 min at 5,000 g
the samples were sonicated for 10 s at 70 volts and incubated for 10 min on ice in lysis buffer. Another centrifugation at 10,000 g separated the supernatant from the pellet and 30 µg of protein of the supernatant was subjected to SDS–PAGE (4–20% gradient) and transferred to Immobilon-P membrane (Millipore) for immunoblotting. The blot was developed with the Supersignal West Pico Chemiluminescent substrate kit or Supersignal West Femto Maximum Sensitivity Substrate kit (Pierce). The antibodies used for immunoblotting were the following: Rabbit anti-human Beclin 1 antibody used for western blotting and immunoprecipitation, was purchased from Cell Signaling Technology. Rabbit c-Myc polyclonal antibody was purchased from Abcam and the rest antibodies used (anti-FYVE-CENT, anti-VPS34, anti-beta-actin, anti-GST and HRP labeled) were described previously 
. For quantitative Western blotting, equal amounts of cell lysates (as measured by protein content) from control and mutant cells were loaded in triplicates on a gel for PAGE. The proteins were transferred to a PVDF membrane and stained with antibodies for FYVE-CENT, Beclin1 and β-actin. The bands were detected using LiCore infrared dye secondary antibodies and the Odyssey imaging system. The bands were quantified using the Odyssey quantifying software.
GST pull-down assay
The GST–FYVE-CENT C-terminus ( amino acid residues 2120–2539), the GST–FYVE-CENT C-terminus (1807–2539) and the GST–FYVE-CENT C-terminus (1807–2539) mutant R1945Q constructs were expressed in BL21 Escherichia coli
, purified and GST-pull down assays were performed as described previously 
Rabbit antibody against FYVE-CENT or rabbit IgG (control) were rotated at RT (room temperature) with Protein A agarose beads for 1 h. Then the beads were washed two times with PBS and two times with 0.2 M triethanolamine, pH 8.2. Crosslinking was performed by rotating the beads in 0.2 M triethanolamine containing 3 mg/ml dimethyl pimelimidate at 4°C overnight. In order to quench the unreacted beads, they were rotated with 10 mM ethanolamine, pH 8.2, at 4°C for 30 min. The beads were washed three times with PBS and were used for immunoprecipitation.
HeLa, HCC-1395 and HCC-1954 cells were grown confluent in 10-cm culture dishes and lysed in ice-cold lysis buffer (20 mM HEPES pH 7.2, 2 mM MgCl2, 100 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100) containing inhibitors (N-ethylmaleimide, mammalian protease inhibitor mixture, phosphatase inhibitor cocktail I and II (Sigma-Aldrich).The lysates were placed on ice and centrifuged at 10,000 g, 4°C and the supernatant was added to the Protein A-coupled magnetic beads (Dynal, Invitrogen) which had been precoupled with rabbit antibody against FYVE-CENT or rabbit IgG as a control, in PBS Tween 20. Antibody coupled magnetic beads and cell lysates were gently mixed for 1 h at 4°C. The beads were then washed with lysis buffer, eluted in 4× sample buffer plus 1 mM DTT at 95°C for 5 min. The eluted proteins were subsequently subjected to SDS–PAGE and immunoblotting as described previously.
All the FYVE-CENT constructs used were generated by PCR with the FYVE-CENT
cDNA (ORF) (NM_015346.2), which was cloned in a pCMV6-XL4 vector by OriGene Technologies, Inc., as template. Synthetic oligonucleotides were from MWG Biotech. The FYVE-CENT R1945Q mutant was prepared by PCR site-directed mutagenesis. PCR errors were excluded by sequencing. For expression as GST fusion proteins in Escherichia coli
BL21 (DE3) cells, the C-terminal part (2120–2539) as well as (1807–2539) and with mutation (R1945Q) of FYVE-CENT were cloned into pGEX-6P-3 (Pharmacia Amersham). The expression plasmid encoding myc-epitope-tagged mouse KIF13A and the Myc-DDK-tagged ORF clone of Homo sapiens TTC19 (NM_017775.2) were obtained as described previously 
. Expression in mammalian cells and purification were performed as described previously 
Assay of rescuing cytokinesis phenotype in RNAi FYVE-CENT depleted cells
HeLa cells were transfected with siRNA (70 nM) against human FYVE-CENT for 72 h. The siRNA-treated cells were then seeded onto coverslips in a 5 cm culture dish and were transfected with myc-tagged C- terminal 1807–2539 and myc-tagged C-terminal 1807–2539 R1945Q FYVE-CENT constructs respectively in three different series of experiments for 36 h. The cells were washed in PBS, stained with anti-myc and anti-α tubulin antibodies and processed in confocal microscopy analysis as described above. The experiment was repeated three times and in total, and 270 back transfected cells were quantified. In parallel, simple depletion experiments using control and FYVE-CENT siRNA were performed in triplicates and quantified using the same stainings and conditions.
RNA interference studies
Single deconvoluted siRNAs against FYVE-CENT (cat.no. D-031136-04), VPS34 (PIK3C3)(cat. no. D-005250-04) and Beclin 1(siRNA 1: cat. no. J-010552-05) were purchased from Dharmacon Research. The siRNA experiments were performed on HeLa cells as described before 
Yeast two-hybrid screening
The yeast two-hybrid screening was based on the C terminus (residues 2120–2539) of FYVE-CENT as bait and performed by Hybrigenics S.A Services using a human T cells RP1 (CEMC7) library.
RNA isolation/cDNA sequencing
Total RNA was isolated from HCC-1395 (control cells) and HCC-1954 (FYVE-CENT R1945Q mutants cells) (1 well of a 6 well plate for each) using the Total RNA Mini Kit (BioRad) according to the manufacturer's descriptions. One microgram RNA was converted to cDNA using the iScript cDNA Synthesis kit (BioRad). Forward and reverse primers, AGGAGGAAAATGAGCTGGTG and CAGCACATCTACCTTGCTGA, were designed with the Primer3 software using default settings, and PCR products were sequenced in forward and reverse using an ABI 3730 DNA Analyzer (Life Technologies).
Values are given as means and s.d in all figures. The p values are calculated based on t-test.