Cell culture and transfection
HUVEC were acquired from PromoCell as cryoconserved pools (C-12203) and cultured on Corning CellBind dishes at 37°C and 5% CO2 in 1:1 mixed medium comprising M199 medium (BioChrom) supplemented with 10% FCS, 30 µg/ml gentamycin, 0.015 µg/ml amphotericin B, and ECGMII (PromoCell) supplemented with 30 µg/ml gentamycin, 0.015 µg/ml amphotericin B. Experiments were conducted with HUVEC passage 3-5.
HUVEC were transfected using the Amaxa nucleofection system (Lonza) according to the manufacturer´s specifications. Per cuvette, cells from a confluent 20-30 cm2 dish together with 1-6 µg plasmid DNA and/or 400 pmol siRNA were resuspended in transfection buffer (4 mM KCl, 10 mM MgCl2, 10 mM sodium succinate, 100 mM NaH2PO4, pH 7.4 adjusted with NaOH). For depletion of proteins using siRNA, HUVEC were transfected with 400 pmol of siRNA twice. 48 h after the first transfection the cells were transfected again with the same amount of the respective siRNA. 24 h after the second transfection cells were subjected to live cell microscopy or lysed for Western blot analysis.
nucleofection system (Lonza) according to the manufacturer´s specifications. Per cuvette, cells from a confluent 20-30 cm2 dish together with 1-6 µg plasmid DNA and/or 400 pmol siRNA were resuspended in transfection buffer (4 mM KCl, 10 mM MgCl2, 10 mM sodium succinate, 100 mM NaH2PO4, pH 7.4 adjusted with NaOH). For depletion of proteins using siRNA, HUVEC were transfected with 400 pmol of siRNA twice. 48 h after the first transfection the cells were transfected again with the same amount of the respective siRNA. 24 h after the second transfection cells were subjected to live cell microscopy or lysed for Western blot analysis.
For live cell microscopy, transfected HUVEC were seeded on 8-chamber µ-slides which were freshly coated with collagen from rat tail (Advanced Biomatrix, 5056) at a concentration of 50 µg/ml in 0.02 M acetic acid solution.
Plasmids and siRNA
A P-selectin construct encoding only the soluble luminal domain of P-selectin, herein referred to as P-sel-lum-mRFP was subcloned from P-selectinLum-mCherry, which was kindly provided by Dan Cutler (MRC Laboratory for Molecular Cell Biology, University College London) 34,35, by introducing the P-selectinLum coding region into the pmRFP1-N1 vector from Addgene (Plasmid No: 54635) via BglII/AgeI restriction sites (courtesy of Nina Criado Santos, Institute of Medical Biochemistry, University of Muenster).
VWF-pHlourin was obtained by replacing mRFP in a VWF-mRFP plasmid, which was kindly provided by Tom Carter (St. George´s University of London, UK) 36,37, with superecliptic pHlourin 38 (courtesy of Anja Biesemann, Institute of Medical Biochemistry, University of Muenster).
Rab27a-EGFP was generated by amplification of the Rab27a cDNA from a HUVEC cDNA library using specific primers that included EcoRI and BamHI restriction sites at the 5’ and 3’ end, respectively, and cloning of the PCR product into the pEGFP-C1 vector (Clontech). Rab27a-mStrawberry was then generated by exchanging the fluorophore of Rab27a-EGFP, using pmStrawberry (Clontech) as the fluorophore source and specific primers with the restriction sites AgeI and BamHI at the 5’ and 3’ end, respectively.
siRNA targeting ATP6V0d1 was obtained from Horizon Discovery (L-019238-02-0010). Rab27a was depleted using the siRNA 5`GGAGAGGUUUCGUAGCUUA-dTdT purchased from Microsynth 28. AllStars Negative Control siRNA was from Qiagen (102781).
Antibodies
For imaging, mouse monoclonal anti-VWF antibodies were acquired from DAKO (M061601-2) and rabbit polyclonal anti-ATP6V1G1 antibodies (16143-1-AP) were obtained from Proteintech. Secondary antibodies (AlexaFluor488, AlexaFluor594) were purchased from Molecular Probes. Rabbit polyclonal anti-ATP6V0d1 antibodies (8274-1-AP) and mouse monoclonal anti-α-tubulin (T5168) were used as primary antibodies in Western blot analysis.
Western blot analysis
For preparation of cell lysates, HUVEC were harvested using trypsin/EDTA. After washing once with PBS, cell pellets were resuspended in 30 µl RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, pH 7.5) supplemented with 1x Complete protease inhibitor cocktail (Roche, 04693116001) per 20 cm2 confluent HUVEC and lysed for 30 min on ice followed by 1 min sonification. Cellular debris was removed by centrifugation at 1250 x g for 10 min at 4°C. Protein loading buffer was added to a final concentration of 1x and the samples were incubated for 10 min at 95°C.
Samples were subjected to 10% SDS-PAGE for 30 min at 70 V and subsequently at 120 V, and blotted onto 0.2 µm nitrocellulose membrane in a wet tank system at 115 V for 1h at 4°C in Tris-Glycine buffer (25 mM Tris, 190 mM glycine, 20% (v/v) methanol). Membranes were blocked using 3% skim milk in TBST (150 mM NaCl, 20 mM Tris-HCl, 0.1 % (v/v) Tween-20, pH 7.4) for at least 30 min and incubated with primary antibodies over night at 4°C. For signal detection, infrared conjugated secondary antibodies (IRdye680RD or IRdye800CW, LICOR) and the Odyssey imaging system (LICOR) were used.
Experiments with HUVEC expressing VWF-pHlourin
Bafilomycin A1 (BafA1) was purchased from Cayman Chemical (Cay11038-1) and dissolved in DMSO. Aliquots of 250 µM were stored at -20°C. Archazolid A (ArchA) was dissolved in DMSO and aliquots of 10 µM were stored at -20°C.
24 h after seeding, transfected HUVEC were incubated with media containing 250 nM BafA1 for 2 h, 0.1% DMSO for 3 h or 40 µM NH4Cl for 3 h at 37°C. Subsequently medium was exchanged with Hank´s Balanced Salt Solution (Sigma, H6648), herein referred to as HBSS, supplemented with 20 mM HEPES pH 7.0-7.6 (Sigma, H0887) and containing 250 mM BafA1, 0.1% DMSO or 40 µM NH4Cl, respectively, and cells were subjected to live cell imaging at 37°C for less than 1 h.
For treatment with ArchA, cells were grown for 24 h on 8-chamber µ-slides following incubation with 2 nM ArchA or 0.02% DMSO for 24 h 39. Subsequently, medium was exchanged with HBSS supplemented with 20 mM HEPES, pH 7.4, and containing 2 nM ArchA or 0.02% DMSO, respectively, and cells were subjected to live cell imaging at 37°C.
For imaging of HUVEC subjected to siRNA treatment (see above), cells were additionally transfected with P-sel-lum-mRFP and VWF-pHlourin together with the second round of siRNA transfection and seeded on 6 cm dishes or 8-chamber µ-slides. Approximately 24 h after the second transfection, cells were lysed for Western blot analysis or analyzed via live cell microscopy at 37°C.
Immunofluorescence stainings
Cells were cultivated on collagen coated coverslips (12 mm diameter) until they reached confluency, then fixed in 4 % PFA in PBS for 10 min at RT and permeabilized using 0.1 % Triton X-100 in PBS for 2 min. Unspecific binding was blocked by addition of 3 % BSA in PBS for at least 30 min, followed by antibody incubation over night at 4°C in 3 % BSA in PBS. Secondary antibodies were diluted 1:200 and incubated for 40 min at room temperature (RT). DAPI staining was conducted with a 0.1 µg/ml solution (Sigma, D9542) for 10 min at RT. After extensive washing, samples were mounted in mounting medium.
Microscopy
Confocal microscopy was performed using an LSM 800 microscope (Carl Zeiss) equipped with a Plan-Apochromat 63×/1.4 oil immersion objective and an Airyscan GaAsP detector.
Image analysis
Confocal images were analyzed using ImageJ. Colocalization analysis was performed by using the plugin JACoP 40. The extent of cooccurrence of P-sel-lum-mRFP with VWF-pHlourin was quantified by calculating Manders coefficient 1 which determines the percentage of total signal from P-sel-lum-mRFP overlapping with signal from VWF-pHlourin 41,42.
Statistics
All statistics were performed using GraphPad PRISM. Asterisks mark statistically significant results: ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Normal distribution was assessed by the Shapiro-Wilk-Test, p < 0.05. Normally distributed data was analyzed employing an unpaired t-test with Welch´s correction. Non-parametric data was analyzed using a Mann-Whitney test.