Supplementary methods
Chemicals, antibodies and reagents
LDL and calcein-AM were purchased from Invitrogen (Karlsruhe, Germany). The
enzyme-linked immunosorbent assay (ELISA) kit for oxidized LDL from human
plasma was from USCN Life Science (Wuhan, China). The kit for measuring total
antioxidant capacity of endothelial cells (ab65329) was from Abcam (Cambridge,
UK). Recombinant human SDF-1α, human SDF-1α quantikine ELISA, monoclonal
mouse anti-SDF-1 (clone 79018 and clone 79014), monoclonal mouse anti-CXCR4
(clones 12G5 and 44716) and monoclonal mouse anti-CXCR7 (clone 11G8) antibody
were from R&D Systems (Wiesbaden, Germany). Polyclonal rabbit anti-CXCR4,
polyclonal rabbit anti-LDL receptor, polyclonal rabbit anti-LDL receptor-related protein
(LRP)-1, polyclonal rabbit anti-37kDa/67kDa laminin receptor and monoclonal mouse
neutralizing anti-laminin receptor (MLuC5) antibody were from Abcam. Phycoerythrin
(PE)-conjugated monoclonal mouse anti-CXCR4 antibody (clone 12G5) and purified
mouse IgG were from BD Biosciences (Heidelberg, Germany). Recombinant human
VEGF165 was obtained from Peprotech (Hamburg, Germany). Monoclonal rabbit antitotal Akt, polyclonal rabbit anti-phospho-Akt (Ser473), monoclonal rabbit anti-total
extracellular signal-regulated kinase (ERK)-1/2, monoclonal rabbit anti-phosphoERK1/2 (Thr202/Tyr204) and polyclonal rabbit anti-β-actin antibody were from Cell
Signaling Technology (Frankfurt, Germany). Monoclonal mouse anti-α-tubulin and
polyclonal rabbit anti-liver X receptor (LXR)-α/β antibody were from Santa Cruz
Biotechnology (Heidelberg, Germany). Monoclonal mouse anti-transferrin receptor
(TFR) antibody was from BD Biosciences (Heidelberg, Germany). Polyclonal rabbit
anti-β-actin, polyclonal rabbit anti-GAPDH antibody and protein G sepharose were
from Sigma (Deisenhofen, Germany), as were the inhibitors of endosomal and
lysosomal protein degradation, bafilomycin A1 and chloroquine diphosphate, and the
LXRα/β inhibitor geranylgeranylpyrophosphate (GGPP). Recombinant human
receptor associated protein (RAP) was from antibodies-online (Aachen, Germany).
Cell surface protein isolation kit was from Thermo Fisher Scientific (Schwerte,
Germany).
Bacterial strains and plasmids
E. coli XL1-blue (Stratagene, Heidelberg, Germany) served as host for plasmid
preparation. Lentiviral vectors pLKO.1 (8453; Addgene, Cambridge, MA, U.S.A.; used
for
control
conditions)
and
pLKO.1-shRNA-CXCR4
(Mission® TRC
shRNA
TRCN0000256864; Sigma, Deisenhofen, Germany; used for CXCR4 knockdown)
were generated in E. coli Stbl3 (Invitrogen). E.coli strains were grown in LB-medium
supplemented with 100 µg/ml ampicillin for plasmid selection.
Lentiviral transduction
For production of recombinant lentivirus, 1×106 HEK 293T cells were co-transfected
with 6 µg of target vector pLKO.1-shRNA, 4 µg of psPAX2 (12260; Addgene) and 2
µg pMD2G-VSVG (12259; Addgene) and incubated for 48 hours. The medium
containing recombinant lentivirus was harvested, filtered through a 0.45 µm filter unit
(Millipore, Schwalbach, Germany) and stored at -80ºC. Functional virus titer was
calculated by transfecting HEK 293T cells with limiting virus dilutions of the green
fluorescent protein (GFP)-carrying vector pWPXL (12257; Addgene) and subsequent
quantification of GFP positive cells by fluorescence microscopy. On average, the viral
titer was 1×106 transduction units/ml.