Ab2900

H1299 cells were seeded at 20,000 cells per well in a 96 well plate (Greiner, 655090). On the second day, cells were treated with DMSO or compounds for 1 h. Then, cells were fixed with 4% PFA and blocked with 10% donkey serum and 0.1% Triton X-100 in 1x DPBS for 1 h. After overnight incubation with anti-EEA1 (Abcam, ab2900, 1:50) at 4 °C, Alexa Fluor 594-conjugated secondary antibody (Invitrogen, A-21207, 1:500) was applied to cells. Nuclei were stained with Hoechst33342 (Thermo Scientific, 62249, 1 ug/mL). Images were taken and analyzed using ImageXpress^® Confocal HT.ai High-Content Imaging System (Molecular Devices, San Jose, CA, USA).

Cells were cultured overnight on poly-D-lysine-coated coverslips in 24 well plates and treated with 200 ng/ml of DOX, encapsulation-DOX, covalent conjugation-DOX, PEG coat-DOX for the indicated time. Cells were fixed with 4% paraformaldehyde in medium overnight at 4 °C. Permeabilization was performed in PBS with 0.1% Triton X-100 for 15 min at RT. After a blocking period of 2 hrs with 1% bovine serum albumin (BSA, Generay Biotech Co., Shanghai, China) in PBS, cells were incubated with EEA-1 (polyclonal, ab2900, Abcam) and mannose 6-phosphate receptor (M6PR, a late endosome marker, 1:1000, ab2733, Abcam) antibodies overnight at 4 °C in the absence of light. After washing twice times with PBS, cells were incubated with Alexa Fluor 405 Goat Anti-Rabbit IgG (H + L) and Alexa Fluor 405 Goat Anti-Mouse IgG (H + L) (1:200 dilution factor, A11008, A21057, Molecular Probes) for 2 hrs at RT. Lastly, cells were mounted, visualized using confocal microscopy LSM700 (Carl Zeiss, Germany) and analyzed using the ZEN software. The number of co-localized (yellow color: co-localized section of DOX and intracellular vesicles) regions was counted in each cells (where a total 10 cells were analyzed for determining the number of EE or LE at a given time).

(Antigen, dilution, vendor, cat. no.): NPC1, 1:500, Abcam, ab134113; Actin, 1:4000, Sigma, A544; LAMP1, 1:100, Developmental Studies Hybridoma Bank University of Iowa, H4A3; Calbindin, 1:500–1:2000, Sigma, 02724; GFAP, 1:500, Dako, z0344; IBA1, 1:250, Abcam, ab5076; NeuN, 1:500, Millipore, abn78; EEA1, 1:400, Abcam, ab2900; F4/80, 1:400, abcam, ab6640.

We used the following primary antibodies for staining: Anti-Stx1 at 1:500 (Synaptic Systems, 110 011), anti-Munc18-1 at 1:500 (Synaptic Systems, 116 002), anti-hDAT at 1:1000 (Sigma-Aldrich, MAB369), anti-hNET at 1:1000 (MAb Technologies, NET17-1), anti-EEA1 at 1:1000 (Abcam, Ab2900) and anti-VMAT2 at 1:4000 (kindly provided by Dr. Garry Miller, Columbia University, New York). Secondary antibodies with conjugated AF568 or AF647 were added in 1:400 dilution.

HTB94 cells (10⁴ cells) were plated on gelatin-coated coverslips in 12-well plates. Wells were washed in serum-free DMEM and incubated for 2 h at 37 °C with TIMP-3, TIMP-3 K26A/K45A, or TIMP-3 K42A/K110A (40 nm) in DMEM with 0.1% FCS. Control wells were incubated without TIMP-3, and LRP dependence was evaluated by incubating wells with RAP (500 nm) for 1 h before the addition of TIMP-3. Wells were washed three times in PBS after each of the steps hereafter. Cells were fixed with 3% paraformaldehyde plus PBS (10 min, 25 °C), blocked with 5% goat serum plus 3% BSA plus PBS (1 h, 25 °C), and permeabilized in 0.1% Triton X-100 plus PBS (15 min, 25 °C). Cells were stained with mouse anti-FLAG M2 (5 μg/ml F1804-5MG, Sigma-Aldrich) and rabbit anti-EEA1 and 0.5 μg/ml ab2900 (AbCam, Cambridge, UK) antibodies in block (3 h, 25 °C). Bound primary antibodies were detected with anti-mouse DyLight 679 (ThermoFisher, Waltham, MA) and anti-rabbit Alexa Fluor 568 (Molecular Probes, Inc., Eugene, OR) in block (1 h, 25 °C). Nuclei were visualized (1 h, 25 °C) with DAPI (ThermoFisher), and actin was visualized with phalloidin Alexa Fluor 488 (Molecular Probes). Cells were mounted in ProLong Diamond antifade mountant (ThermoFisher) and analyzed on a PerkinElmer Life Sciences spinning disc confocal microscope equipped with a Nikon TE-2000 CCD camera (×40 objective lens).