Fig 1: Expression of endothelial-specific markers and FcRn in established BOEC lines. (A–D) Confluent monolayers of BOECs were prepared for immunofluorescence microscopy and flow cytometry, as described in the Materials and Methods. (A,B) BOECs were stained with either CD144 (red) (A) or intracellular vWF (green) (B), and shown are confocal images (left) and FACS analysis (right). White arrows (A) indicate CD144 at the PM. Cells were gated on single, live cells and the histograms are shown for CD144-specific (red) and vWF-specific (green) fluorescence signals. Control cells were stained using the respective secondary antibodies only (grey). Scale bars: 10 µm. (C,D) Monolayers of BOECs were fixed with PFA (C) or methanol (D) and either stained for CD31 (green) (C) or vWF (green) (D) and CD144 (red). Cell nuclei were visualised using DAPI. White arrows indicate intracellular junctions between single BOECs. Scale bars: 10 µm. (E) Cultured BOECs and BMDMs isolated from FcRn KO, hFcRnTg/Tg line 276 or hFcRnTg/Tg line 32 mice were analysed by immunoblotting, with antibodies against human FcRn or GAPDH after stripping the membrane, and HRP-conjugated secondary antibodies. Chemiluminescence was detected using a ChemiDoc system (Bio-Rad). Data have been consolidated from the same immunoblot. The original uncropped blot is shown in Fig. S7. (F) BOECs were fixed with TCA, permeabilised with 0.1% Triton-X-100 and stained for human FcRn (red), and nuclei were visualised with DAPI (blue). White arrows indicated enlarged FcRn-positive endosomal structures. Scale bars: 10 µm (left); 5 µm (right). (G,H) BOECs were fixed with TCA (G) and methanol (H), and stained for human FcRn (red), either EEA1 (green, G) or CD63 (green, H), and DAPI (blue). In G, white arrows indicate the enlarged FcRn- and EEA1- positive endosomal structures. Scale bars: 10 µm (merge); 2 µm (zoom). Images are representative of more than three independent experiments.
Fig 2: Intracellular trafficking of HSA–AF488 in FcRn–mCherry-transduced live BOECs. (A–C) BOECs transduced with FcRn–mCherry (red) were pulsed with HSA–AF488 (green) for 15 min at 37°C and the fluorescence signal was chased for 40 min in live cells. Confocal microscopy images of live cells were taken immediately after the pulse (0 min) (A) or at 5, 20 (A), 25 (B) or 40 (C) min chase. In B, the field from 20 min in A was monitored for the 25–28 min chase time, and zoomed images of ROIs are shown. In C, shown is the maximum-intensity projection of z-stack optical sections. White arrows indicate HSA–AF488- and FcRn–mCherry-double-positive endosomal structures. White asterisks (A) indicate HSA–AF488-positive tubular transport carriers. Sky blue arrows (C) indicate tubular carriers that were negative or only faintly positive for HSA–AF488. Cell boundaries are indicated by dotted lines. Images are representative of more than three independent experiments. Scale bars: 10 µm (merge); 5 µm (zoom).
Fig 3: HSAH464Q–AF488 is excluded from tubular carriers in FcRn–mCherry-transduced live BOECs. (A,B) BOECs transduced with FcRn–mCherry (red) were pulsed with HSAH464Q–AF488 (Mutant–AF488, green) for 15 min at 37°C and the fluorescence signal chased for 40 min in live cells. The same field is shown throughout the chase in the live cells. Confocal images of live cells after 25, 27, 28 and 30 min chase (A) and after 40 min chase (B) are shown. White arrows indicate FcRn–mCherry-positive tubular transport carriers. (C) BOECs were pulsed with both HSAH464Q–AF488 (Mutant–AF488, green) and wildtype HSA–AF568 (red) for 15 min at 37°C, and the fluorescence signals chased for 40 min in live cells. Two ROIs were defined to follow the itinerary of single tubular transport carriers during the 32–40 min chase, as indicated. White arrows indicate wildtype HSA–AF568-positive tubular transport carriers. Cell boundaries are indicated by dotted lines. Images are representative of more than three independent experiments. Scale bars: 10 µm (merge); 5 µm (zoom).
Fig 4: FcRn–mCherry does not reside in the recycling endosomes in transduced live BOECs. (A) BOECs were transfected with Rab11–GFP (green) using Lipofectamine 3000. After 48 h, cells were pulsed for 15 min with Alexa Fluor 568-labelled transferrin (Tf–AF568, red) at 37°C. (B) BOECs were transduced with 10 µl lentivirus containing pFUGW-B2M-FcRn_mCherry (red) for 24 h, and the monolayers were washed and incubated for an additional 24 h. Transduced cells were pulsed for 15 min with Tf–AF488 (green) at 37°C. (A,B) Monolayers of BOECs were washed and live cells imaged at 37°C and 5% CO2 with a FV3000 Olympus confocal microscope. White arrows indicate low level of overlap in the perinuclear region. Images represent maximum projections of whole-cell z-stacks from two independent experiments. Scale bars: 10 µm (original images); 5 µm (zoomed images).
Fig 5: Pulse-chase of internalised HSA and HSAH464Q with endosomal and/or lysosomal markers in fixed BOECs. (A–D) BOECs were pulsed with either HSA–AF488 (green) (A–C) or non-FcRn-binding HSAH464Q (Mutant–AF488, green) (D) for 30 min at 37°C. Monolayers were washed and the intracellular fluorescence signal chased for up to 60 min at 37°C. After the chase periods, cells were directly fixed with PFA and stained for EEA1 (red; A,B) or CD63 (red; C,D). Nuclei were visualised using DAPI (blue). Images represent maximum projections of whole-cell z-stacks from more than three independent experiments. Scale bars: 10 µm. (B) Quantification of co-localisation by Manders' correlation coefficient M1. The parameters were calculated using ImageJ and values shown for calculated parameters above threshold; n=5 cells from one experiment. Data were analysed using Fisher's LSD test. Error bars represent s.e.m. **P<0.01; ***P<0.001; ****P<0.0001. (C,D) The profiles of two line scans per image are shown for both fluorophores. The location of the respective line scans is indicated by white lines. Images represent maximum projections of whole-cell z-stacks. Data are representative of more than three independent experiments. Scale bars: 10 µm.
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