Journal: The Journal of Cell Biology

Article Title: The glycoprotein GP130 governs the surface presentation of the G protein–coupled receptor APLNR

doi: 10.1083/jcb.202004114

Figure Lengend Snippet: 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.

Article Snippet: Total protein lysates from GP130- and/or APLNR-transfected HEK293T cells were denaturated (55°C, 10 min) and treated overnight with EndoH and for 1 h with PGNase (New England Biolabs) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Confocal Microscopy, Software, Immunoprecipitation, Western Blot, Proximity Ligation Assay, Transfection, Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: The glycoprotein GP130 governs the surface presentation of the G protein–coupled receptor APLNR

doi: 10.1083/jcb.202004114

Figure Lengend Snippet: GP130 does not affect all APLN/APLNR signaling activities. (A) S6 phosphorylation was monitored by flow cytometry in WT (blue) and GP130 KO#2 (green) patient-derived GSCs (mesenchymal GSC#1) cultured in MF conditions and MF containing APLN only (1 µM). Isotype staining is also shown as a control for gating. Histogram represents the mean ± SEM percentage of phospho S6 ribosomal protein (pS6)–positive cells in two independent experiments. SSC, side scatter. (B) A radioligand binding assay of APLN to APLNR was measured in the presence of the either competitive APLNR antagonist (MM54, black) or anti-GP130 antibodies (blue) in APLNR–stably expressing cells. Data are expressed as the mean ± SEM percentage of binding inhibition on technical duplicates. (C) Patient-derived GSCs (mesenchymal GSC#1) transfected with nonsilencing (sic, black) and either GP130 or APLNR targeting siRNA duplexes (si GP130 , blue and si APLNR , green) were analyzed by qPCR for APLNR and GP130 . Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (D) qPCR was performed in WT (blue) and GP130 KO (#2, green; and #7, orange) GSC#1. Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (E) HEK293T cells were transfected with GP130 and/or APLNR encoding plasmids. Protein lysates were incubated with buffer, EndoH or PGNase, and further analyzed by Western blot, as indicated. All data are representative of at least three independent experiments. ***, P < 0.001 using ANOVA tests.

Article Snippet: Total protein lysates from GP130- and/or APLNR-transfected HEK293T cells were denaturated (55°C, 10 min) and treated overnight with EndoH and for 1 h with PGNase (New England Biolabs) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Staining, Radio Ligand Binding Assay, Stable Transfection, Expressing, Binding Assay, Inhibition, Transfection, Incubation, Western Blot

Journal: Scientific Reports

Article Title: Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures

doi: 10.1038/s41598-022-14875-7

Figure Lengend Snippet: Sequence alignments of ( a ) WNT3 protein for mouse, human and chickens. Chicken vs mouse and chicken vs human are 96% identical (human vs mouse: 98%). ( b) Alignment of RSPO1 for mouse, human and chicken. Chicken and mouse is 65% identical and chicken and human is 68% (human and mouse: 89%). Non-consensus amino acids (grey).

Article Snippet: Basic culture medium, supplemented with 1:5 of supernatant of Rspo1 expressing Hek293t cells (Sigma-Aldrich; SCC111), 1:5 of supernatant of L Wnt-3A cells (ATCC, Molsheim, France; CRL-2647), and the BMP4 inhibitor DMH1 1.3 µM (Sigma-Aldrich; D8946).

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures

doi: 10.1038/s41598-022-14875-7

Figure Lengend Snippet: Growth factors RSPO1 and WNT3, from murine or chicken origin, are important for establishing intestinal organoid growth. (a) Immunoblot analysis of chicken RSPO1 and chicken WNT3 protein expression in transfected Hek293t cells. Actin was used as the protein loading control and anti-flag HRP was used to visualize RSPO1 and WNT3. Empty Vector (E.V.) is taken along as a control. (b) Brightfield images of mouse intestinal organoids (I,II) and chicken intestinal organoids (III,IV) at passage 2 (5 days after passage). Cultured in Matrigel with ICM, supplemented with Rspo1 and Wnt3 of murine origin, (c) or supplemented with chicken-derived RSPO1 and WNT3. Images and immunoblot are representative of three independent cultures. Scale bar 200 µm.

Article Snippet: Basic culture medium, supplemented with 1:5 of supernatant of Rspo1 expressing Hek293t cells (Sigma-Aldrich; SCC111), 1:5 of supernatant of L Wnt-3A cells (ATCC, Molsheim, France; CRL-2647), and the BMP4 inhibitor DMH1 1.3 µM (Sigma-Aldrich; D8946).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Cell Culture, Derivative Assay

Journal: Scientific Reports

Article Title: Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures

doi: 10.1038/s41598-022-14875-7

Figure Lengend Snippet: Supplementary compounds for organoid growth. Brightfield images of embryonic chicken organoid cultured in Matrigel with BCM of passage 1, 1,2 and 3 days after splitting. ( a,e,i) with additional RSPO1 and WNT3 of chicken origin. ( b,f,j) with additional RSPO1/WNT3 of chicken origin and 0.2 µM Forkhead box O1-inhibitor (FOXO1-i). ( c,g,k) with additional RSPO1/WNT3 and 2.5 µg/ml prostaglandin E2 (PGE2) ( d,h,l) . With all 4 supplemented growth factors (WNT3/RSPO1/PGE2/FoxO1-i). Images are representative of three independent cultures. Scale bar: 1000 µm. (m) Diameter of organoids when culturing with different supplements, were measured manually and plotted in Graphpad Prism. Four outliers were excluded after performing an outlier test in Graphpad Prism. Statistics were performed using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. *p < 0.05 **p < 0.01.

Article Snippet: Basic culture medium, supplemented with 1:5 of supernatant of Rspo1 expressing Hek293t cells (Sigma-Aldrich; SCC111), 1:5 of supernatant of L Wnt-3A cells (ATCC, Molsheim, France; CRL-2647), and the BMP4 inhibitor DMH1 1.3 µM (Sigma-Aldrich; D8946).

Techniques: Cell Culture