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

Figures

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  Figure S1.. JAK inhibitors impaired APLN-based GSC maintenance. (A) Patient-derived GSCs (mesenchymal GSC#1) were cultured in MF conditions (green) or MF containing APLN (1 µM) plus either vehicle (APLN+DMSO, black) or ruxolitinib (APLN+Ruxo, 1 µM, pink). Linear regression plot of in vitro LDA is shown. Alternatively, LDA assays were done in mitogen-containing medium plus vehicle (NS34+DMSO, black) and NS34 plus ruxolitinib (NS34+Ruxo, pink). Data are representative of two independent experiments. (B) Tumorspheres per FOV were manually, single-blindly counted in similarly treated cells cultured in MF medium, mitogen-containing medium (NS34), or MF containing APLN (1 µM, APLN). DMSO (black) and the JAK inhibitors ruxolitinib (1 µM, red) and filgotinib (1 µM, blue) were also added as indicated. Data are presented as the mean ± SEM from three independent experiments. Each dot (n > 25) represents one sample count. All data are representative of at least three independent experiments. ***, P < 0.001 using ANOVA tests.

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  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…

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  Figure S2.. Effects of nonionic detergents on APLNR localization. GSC#1 was fixed (PBS) and further permeabilized (Triton, saponin, and digitonin) before analysis by confocal microscopy for APLNR (red) and nuclei (DAPI, blue). Scale bars, 10 µm. All data are representative of at least three independent experiments.

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  Figure 2.. GP130 is required for APLN-mediated GSC expansion. (A) Patient-derived GSCs (mesenchymal GSC#1, mesenchymal GSC#4, and classical GSC#9) were cultured in MF conditions (green), MF medium containing APLN (1 µM) plus control isotype Ig (2 µg/µl, black), and MF containing APLN (1 µM) plus anti-GP130 antibody (2 µg/µl, red). Linear regression plot of in vitro LDA is shown for GSC#1. Data are representative of two independent experiments. Stemness frequency was calculated from LDA in GSC#1, GSC#4, and GSC#9. Data are presented as the mean ± SEM on two independent experiments. (B) Tumorspheres per FOV were manually, single-blindly counted in GSC#1, GSC#4, and GSC#9 treated as in A. Data are presented as the mean ± SEM of three independent experiments. Each dot (n > 25) represents one sample count. (C) Silencing efficiency is shown by Western blot for GSC#1 and GSC#4 transfected with nonsilencing (sic, black) or GP130 targeting siRNA duplexes (siGP130, blue). (D) Tumorspheres per FOV were manually, single-blindly counted in GSC#1 and GSC#9, as treated in C, in MF medium, in mitogen-containing medium (NS34) and MF containing APLN (1 µM, APLN). Data are presented as the mean ± SEM of three…

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  Figure S3.. 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 (siGP130, blue and siAPLNR, 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)…

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  Figure 3.. GP130 contributes to APLNR availability at the plasma membrane. (A) Flow cytometry analysis of APLNR and GP130 in patient-derived GSCs (mesenchymal GSC#1, mesenchymal GSC#4, and classical GSC#9) transfected with nonsilencing (sic, blue) and GP130 targeting siRNA duplexes (siGP130, green). Ig control staining plots are shown (red). Histograms present the mean fluorescence intensity (MFI) normalized to respective sic conditions for GP130 and APLNR staining as indicated. Data are presented as the mean ± SEM of three independent experiments. (B) Flow cytometry analysis of APLNR and GP130 in GSC#1 transfected with nonsilencing (sic, blue) and APLNR targeting siRNA duplexes (siAPLNR, green). Ig control staining plots are shown (Ig, red). Data are representative of three independent experiments. (C) Flow cytometry analysis of GP130, APLNR and JAMC in WT (blue) and GP130 KO (#2, green, and #7, orange) GSC#1. Ig control staining plots are shown (Ig, red). Data are representative of three independent experiments. (D) Flow cytometry analysis of GP130 and APLNR in GSC#1 WT (parental, blue), GP130 KO (#2, green, and #7, orange), and GP130 KO reconstituted with GP130 cDNA (#2+GP130, light…

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  Figure 4.. ELMOD1 is modulated by GP130 expression. (A) RNA was extracted from three independent batches of WT (blue) and GP130 KO (#2, green; and #7, orange) patient-derived GSCs (mesenchymal GSC#1) and analyzed by RNA-seq. Representation of principal-component (PC) analysis and nonsupervised clustering show are shown. “Up” (orange) and “down” (blue) differentially expressed genes are also represented by pairs. n (226, 174, and 22) indicates the number of differentially expressed genes for each comparison. (B) qPCR analysis of up- and down-regulated hits in WT (blue) and GP130 KO#2 (green) GSC#1. Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (C) Confocal analysis of ELMOD1 (green) and nuclei (DAPI, blue) in WT and GP130 KO (#2 and #7). Data are representative of three independent experiments. Scale bars, 10 µm. (D) qPCR analysis of ELMOD1, ELMOD2, and ELMOD3 in WT (blue) and GP130 KO (#2, green; and #7, orange). Data are presented as the mean ± SEM fold change on three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (E) qPCR analysis of ELMOD1 in GSC#1…

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  Figure S4.. Prediction of STAT3 consensus binding sites on the three loci surrounding ELMOD1, ELMOD2, and ELMOD3 genes. Putative STAT3 consensus binding sites were predicted using JASPAR and ENCODE (transcription factor ChIP-seq database), in a 500-kb region on chromosome (chr) 11, 4, and 2, surrounding ELMOD1, ELMOD2, and ELMOD3 genes, respectively.

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  Figure 5.. ELMOD1 is involved in the GP130-dependent trafficking of APLNR. (A) qPCR analysis of ELMOD1 in GSC#1 transfected with nonsilencing (sic, dark blue) and three independent ELMOD1 targeting siRNA duplexes (siD1.1, siD1.2, and siD1.3, in green, orange, and light blue, respectively). Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (B) Flow cytometry analysis of APLNR and GP130 in sic (dark blue) and siELMOD1 (seq.1, 0.2, and 0.3, in green, orange, and light blue, respectively). Ig control staining plots are shown (red). Data are representative of at least three independent experiments. Heatmap recapitulates the mean MFI for GP130 and APLNR staining as indicated, normalized to sic, in three independent experiments. (C) Flow cytometry analysis of anti-APLNR uptake in sic (blue) and siELMOD1 (green) GSC#1 upon 15-min incubation at 37°C. Ig control staining plots are shown (red). Boxplots depict the uptake of the anti-APLNR uptake at the indicated time points (15 min) following 37°C incubation. Line delineates the mean, and boxes show upper and lower quartiles. Data are representative of four…

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  Figure S5.. Modulation of APLNR membrane expression by ARF-related GEFs. (A) qPCR analysis of ARFs1-6 and ARLs1-2 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. (B) qPCR analysis of the indicated targets in GSC#1 transfected with nonsilencing (sic) and indicated siRNA duplexes. Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (C) qPCR analysis of ARNO (CYTH2) and BRAG2 (IQSEC1) GSC#1 transfected with nonsilencing (sic) and respective siRNA duplexes. Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (D) Flow cytometry analysis of APLNR in WT (blue) and GP130 KO#2 cells transfected with sic (blue) and ARNO and BRAG2 siRNA duplexes (green and orange, respectively). Ig control staining plots are shown (red). Histograms present the MFI normalized to respective sic conditions for APLNR staining as indicated. Data are presented as the mean ± SEM of three…

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  Figure 6.. ARF GTPases controls APLNR availability at the plasma membrane. (A) Flow cytometry analysis of APLNR in sic (blue) and siRNA duplexes (green) targeting the indicated ARFs. Ig control staining plots are shown (red). Histograms present the MFI normalized to respective sic conditions for APLNR staining in the indicated ARF/ARL-silenced cells. Data are presented as the mean ± SEM of three independent experiments. (B) qPCR analysis of ARF3 in GSC#1 transfected with nonsilencing (sic, dark blue) and ARF3 targeting siRNA duplexes (seq.1, 0.2, and 0.3, in green, orange, and light blue, respectively). Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (C) Flow cytometry analysis of APLNR in sic (blue) and siARF3 (seq.1, 0.2, and 0.3, in green, orange, and blue, respectively) in WT and GP130 KO#2. Graph recapitulates the normalized MFI for APLNR staining as indicated in three independent experiments. (D) Boxplots depict the internalization and recycling of the anti-APLNR in sic (blue) and siARF3 (green) WT and GP130 KO#2 cells at 15-min pulse time (iAPLNR), followed by a 60-min chasing at 37°C…

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  Figure S6.. ARF GTPases contribute to APLNR availability at the plasma membrane. (A) qPCR analysis of ARF6 in GSC#1 transfected with nonsilencing (sic, dark blue) and three independent ARF6 targeting siRNA duplexes (si#1, si#2, and si#3, in green, orange, and light blue, respectively). Data are presented as the mean ± SEM fold change of three independent experiments using ACTB and HPRT1 as housekeeping genes for normalization. (B) Flow cytometry analysis of APLNR in sic (blue) and siARF6 (seq#1, #2, and #3, in green, orange, and blue, respectively). Ig control staining plots are shown (red). Histograms present the MFI normalized to respective sic conditions for APLNR staining as indicated. Data are presented as the mean ± SEM of three independent experiments. (C) Boxplots depict the uptake of the anti-APLNR uptake, in sic (blue) and siARF6 (green) GSC#1, at the indicated time points (15 min) following 37°C incubation. Line delineates the mean, and boxes show upper and lower quartiles. Data are representative of four independent experiments. Flow cytometry analysis of anti-APLNR uptake in sic (blue) and siARF6 (green) GSC#1 upon 15-min incubation at 37°C. Ig control staining plots are shown…