Figure 1. APLNR interacts with GP130 at... | Rockefeller University Press

$APLNR interacts with GP130 at membrane microdomains at the surface of GSCs. (A) Patient-derived GSCs (mesenchymal GSC#1) were fixed, permeabilized and analyzed by confocal microscopy for GP130 (green) and APLNR (red). Merge images are shown, colocalization were visualized using Fiji software. Scale bars, 10 µm. Pearson’s correlation factor was measured using the Fiji Coloc2 analysis tool in >50 cells from at least three independent experiments. Data are presented as violin plot, and dashed line delineates the mean. (B) Protein lysates from patient-derived GSC#1 were processed from immunoprecipitation using control isotype Ig (black) or anti-APLNR antibody (blue). Samples were resolved on Tris-glycine SDS gels for Western blot using the indicated antibodies. Densitometry analysis was performed in three independent experiments and expressed as the mean fold change ± SEM. (C) Proximity ligation assay (PLA) was performed in GSC#1 using either APLNR alone (Ig/APLNR) or APLNR/GP130 antibodies. PLA signal is indicated in red. Nuclei are shown in blue (DAPI). Scale bars, 10 µm. The number of dots per cell was counted in single-blinded images from three independent experiments (n = 63 in Ig, n = 145 in GP130). Dashed lines delineate the mean. (D) HEK293T cells were transfected with GP130 and/or APLNR and then fixed, permeabilized, and analyzed by confocal microscopy for GP130 (green) and APLNR (red). Merge images are shown. Images are representative of three independent experiments. Scale bars, 10 µm. Similarly, protein lysates from transfected cells were processed for immunoprecipitation with APLNR (blue) or GP130 (pink) antibodies. IP fractions were analyzed by Western blot, as indicated. Densitometry analysis was performed in three independent experiments and expressed as the mean fold change ± SEM. (E) GSC#1 were fixed (PBS) and further permeabilized (Triton) before analysis by confocal microscopy for APLNR (red) and nuclei (DAPI, blue). Alternatively, cells were pretreated with vehicle (DMSO 1%, 30 min) and MBCD (10 mM, 30 min). Scale bars, 10 µm. (F) GSC#1 cells were fixed and analyzed by confocal microscopy for GPI-enriched domains (FLAER, gray), GP130 (green), and APLNR (red). Merge images are shown. Scale bars, 10 µm. (G) Structured illumination microscopy was deployed to image nanoclusters of APLNR and GP130 in superresolution. Scale bars, 1 µm. Graph shows the quantification of staining intensity alongside the white broken line in the merge panel. All data are representative of at least three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 using ANOVA tests. IP, immunoprecipitation.$

Figure 1.

APLNR interacts with GP130 at membrane microdomains at the surface of GSCs. (A) Patient-derived GSCs (mesenchymal GSC#1) were fixed, permeabilized and analyzed by confocal microscopy for GP130 (green) and APLNR (red). Merge images are shown, colocalization were visualized using Fiji software. Scale bars, 10 µm. Pearson’s correlation factor was measured using the Fiji Coloc2 analysis tool in >50 cells from at least three independent experiments. Data are presented as violin plot, and dashed line delineates the mean. (B) Protein lysates from patient-derived GSC#1 were processed from immunoprecipitation using control isotype Ig (black) or anti-APLNR antibody (blue). Samples were resolved on Tris-glycine SDS gels for Western blot using the indicated antibodies. Densitometry analysis was performed in three independent experiments and expressed as the mean fold change ± SEM. (C) Proximity ligation assay (PLA) was performed in GSC#1 using either APLNR alone (Ig/APLNR) or APLNR/GP130 antibodies. PLA signal is indicated in red. Nuclei are shown in blue (DAPI). Scale bars, 10 µm. The number of dots per cell was counted in single-blinded images from three independent experiments (n = 63 in Ig, n = 145 in GP130). Dashed lines delineate the mean. (D) HEK293T cells were transfected with GP130 and/or APLNR and then fixed, permeabilized, and analyzed by confocal microscopy for GP130 (green) and APLNR (red). Merge images are shown. Images are representative of three independent experiments. Scale bars, 10 µm. Similarly, protein lysates from transfected cells were processed for immunoprecipitation with APLNR (blue) or GP130 (pink) antibodies. IP fractions were analyzed by Western blot, as indicated. Densitometry analysis was performed in three independent experiments and expressed as the mean fold change ± SEM. (E) GSC#1 were fixed (PBS) and further permeabilized (Triton) before analysis by confocal microscopy for APLNR (red) and nuclei (DAPI, blue). Alternatively, cells were pretreated with vehicle (DMSO 1%, 30 min) and MBCD (10 mM, 30 min). Scale bars, 10 µm. (F) GSC#1 cells were fixed and analyzed by confocal microscopy for GPI-enriched domains (FLAER, gray), GP130 (green), and APLNR (red). Merge images are shown. Scale bars, 10 µm. (G) Structured illumination microscopy was deployed to image nanoclusters of APLNR and GP130 in superresolution. Scale bars, 1 µm. Graph shows the quantification of staining intensity alongside the white broken line in the merge panel. All data are representative of at least three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 using ANOVA tests. IP, immunoprecipitation.

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