Pitstop 2

Two inhibitors of NP internalization were used: Dynasore (ab120192, Abcam) and Pitstop2 (SML-1169, Sigma-Aldrich). Both inhibitors were used at a concentration of 40 μM/dish and they remained active 3 h prior to NP treatment. Dynasore inhibits dynamin, an important factor in receptor-mediated endocytosis, while Pitstop2 blocks clathrin-mediated endocytosis in the receptor-mediated endocytosis pathway. Both inhibitors were added to MSCs for 30 min before treatment with positively charged PCS NPs. CID 1067700 (cat. 2184, Axon Medchem, Groningen, Netherlands) was used to block Rab7 by competitive inhibition.

All experiments were conducted in accordance with the German federal law and the EU directive 2010/63. The protocols were approved by the local authority (Regierungspräsidium Karlsruhe). Sprague Dawley rats of either sex were injected at P12/13 with either HRP only (10 mg/ml in PBS, SERVA, Heidelberg, Germany), HRP supplemented with 1% DMSO or HRP along with Pitstop-2 (120 μM, Sigma) into the MNTB as described previously (Körber et al., 2012 (link)). In brief, rats were anesthetized, transferred into a non-traumatic stereotaxic frame (Kopf Instruments, Tujunga, CA) and 2 μl of marker or drug solution were injected to the following coordinates relative to bregma and midline (x, y, z in mm): 0.95, –6.4, –7.6. Pitstop-2 was delivered in a two-step injection. First, Pitstop-2 was administered to the MNTB alone and only in the second injection, 15 min after the first one, Pitstop-2 was co-injected with HRP to the identical coordinates. The rats remained anesthetized between the two injections. After surgery, rats recovered quickly and behaved normally while they were exposed to standard laboratory environmental noise (radio, air conditioner, human conversation) for 30 min.

HUVECs were pretreated with SU‐1498 (SML1193, Sigma Aldrich) at 20 μM for 4 h or Hydroxy‐Dynasore (SML0340, Sigma Aldrich) at 100 μM or Pitstop‐2 (SML1169, Sigma Aldrich) at 30 μM for 1 h. The resulting cells were exposed to 15 dyn/cm² of shear stress for 5, 10 and 30 min. The cells were fixed and analyzed by immunofluorescence staining for VE‐PTP and VE‐cadherin.

Human embryonic kidney reporter cell lines (HEK-Blue hTLR2; 5x10⁴ cells/well) were infected or treated as described in previous sections in a 96-well flat bottom plate for 24h at 37°C, 5% CO2 atmosphere. To test if endocytosis is required for NF-κB stimulation, wortmannin (Sigma Aldrich), pitstop 2 (Sigma Aldrich), dynasore (Sigma Aldrich) or ammonium chloride (NH₄Cl, Merck) were added 1h before infection or treatment with the agonist. After 24h post infection or treatment, analysis of NF-κB stimulation was assessed by mixing the supernatants with QUANTI-Blue (InvivoGen). Absorbance was measured at 630 nm using Synergy HT multi-mode microplate reader (BIOTEK). PAM3CSK4 (50 ng/mL) was used as a positive control.

Monensin, methyl-β-cyclodextrin, cytochalasin D, Pitstop-2, and EIPA were purchased from Sigma. Dynasore and MiTMAB were purchased from Calbiochem. Bafilomycin A1 was purchased from ApexBio. Ammonium chloride was purchased from Fisher Scientific. Dyngo-4a was purchased from Abcam. NSC23766 was purchased from Santa Cruz Biotechnology.

Reagents for preparation of chemically defined medium (CDM) were purchased from VWR and Sigma. The medium was prepared as previously described (van de Rijn and Kessler, 1980 (link)). Todd-Hewitt broth, yeast extract, paraformaldehyde (PFA), nocodazole, Pitstop 2, Wortmannin, and Dynasore were purchased from Sigma. RPMI 1640 medium, Fetal bovine serum (FBS), Trypsin and other cell culture reagents were purchased from Hyclone, Thermo-Fisher. Gentamicin, AlexaFluor 647-conjugated donkey anti-goat antibody, AlexaFluor 568-conjugated phalloidin, AlexaFluor 488-conjugated transferrin or cholera toxin subunit B, and Fluorescein-conjugated dextran 70,000 MW were purchased from Invitrogen, Thermo-Fisher Scientific. Cytochalasin D was purchased from Calbiochem. Nystatin was purchased from Alfa Aesar. Anti-GAS antibody was purchased from Abcam.

Hoechst 33342 and Pitstop 2 were purchased from Sigma-Aldrich. Pitstop 2 were dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions. The lipophilic dyes DiO and DiD were purchased from Biotium. The fluorescent dyes Alexa Fluor 647 and Alexa Fluor 488 phalloidin were purchased from Invitrogen. anti-β-tubulin was purchased from Abcam (USA). peroxidase-conjugated affinipure goat anti-rabbit IgG were purchased from proteintech (USA).
Using the primers listed in Table 1, the full-length CLCs were constructed in vectors including pcDNA3.1-flag, pEGFP-N3, and pmDsRed-C1 (Invitrogen). Site-directed mutants, including EaCLCa-W119R and EaCLCb-W122R, were all subcloned into the pEGFP-N3, pmDsRed-C1 and pcDNA3.1-flag vectors using specific primers (Table 1) and the Fast Mutagenesis Kit V2 (Vazyme). Tryptophan (W)119 of CLCa and W122 of CLCb were both replaced with arginine(R). In addition, the pEGFP-Rab5 vector was maintained in our laboratory. The constructed plasmids were confirmed by sequencing.