Plasmid mega prep kit

DOTAP, DOPC and DOPE-PEG2000 were purchased from Avanti Polar lipids as chloroform solutions. MVL5 was synthesized as described previously [39 (link)]. The fluorescent lipids TRITC-DHPE and Texas Red-DHPE (Invitrogen, Carlsbad, California) have excitation and emission maxima of 555/589 nm and 580/615 nm, respectively. The RGD-PEG2K-lipid contained a GRGDSP sequence covalently bound to the distal end of PEG2K. It was custom synthesized on solid phase using Fmoc-amino acids and a lipid-PEG2K acid. The pGL3-control vector coding the Luciferase gene (Promega, Fitchburg, Wisconsin) was propagated in E. coli, and purified using a Qiagen Plasmid Mega Prep Kit. The GFP-Rab5-Q79L plasmid was a gift from the Weimbs lab (UCSB) and propagated and purified as decribed above for pGL3. For cell imaging studies, the pGL3 vector was labeled using the Mirus Bio Label IT Nucleic Acid Labeling Kit with Cy5 (excitation/emission maximum: 649 nm/670 nm) according to the manufacturer’s protocol. For labeling of early endosomes, the CellLights Early Endosome-GFP BacMam 2.0 (Life Technologies, Carlsbad, California) reagent was used according to manufacturer’s protocol.

Lipid stock solutions of DOPC and DOTAP in chloroform were purchased from Avanti Polar Lipids. PTXL was purchased from Acros Organics and dissolved in chloroform at 10.0 mM concentration. CellTiter 96® AQueous-One Solution Cell Proliferation Assay was obtained from Promega. Paclitaxel–Oregon Green® 488 Conjugate, Texas Red–DHPE, and glycerol monooleate (GMO) were purchased from Thermo Fisher Scientific as powders and dissolved in chloroform to 190 μM, 81 μM and 10 mM concentrations, respectively. Custom DNA oligomers (sense strand: ACGCTTT; antisense strand: AGCGTTT) were purchased from Sigma-Aldrich. Luciferase plasmid pGL3 (purchased from Promega) was propagated in Escherichia coli and purified using a Qiagen Plasmid Mega Prep Kit.

DOTAP, DOPC and DOPE-PEG2000 (referred to here as PEG2K-lipid) were purchased as solutions in chloroform from Avanti Polar Lipids (Alabaster, AL). The RGD-PEG2K-lipid contains a GRGDSP peptide (Gly-Arg-Gly-Asp-Ser-Pro-OH) covalently attached to the distal end of the PEG-chain of a custom PEG2000-lipid. It was synthesized via Fmoc solid phase synthesis, employing a lipid-PEG-acid building block in the final coupling step. The chemical structures of the lipids are shown in the Supplementary Material (Fig. S2). TRITC-DHPE (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine) was purchased from Invitrogen and has an excitation and emission maximum of 555 nm and 580 nm, respectively. The luciferase plasmid (pGL3) used in transfection experiments was purchased from Promega. The GFP-tubulin (Clontech) and pGL3 plasmids were propagated in E. coli and purified using a Qiagen Plasmid Mega Prep Kit. For live-cell imaging studies, the pGL3 vector was labeled using the Mirus Bio Label IT Nucleic Acid Labeling Kit with Cy5 (excitation/emission maximum: 649 nm/670 nm) according to the manufacturer’s protocol.