Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2021 Jul 21;220(9):e202004114. doi: 10.1083/jcb.202004114

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2021 Trillet et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

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 independent experiments. Each dot (n > 25) represents one sample count. (E) Schematic representation of the GP130 (IL6ST) gene, with positioning of the CRISPR sequence guide at the junction between intro and exon8 (underlined sequence), that encodes for the N-terminal part of the extracellular domain. Genomic sequences in WT and two bi-allelic clones (KO#2 and #7) are shown. TM, transmembrane domain. (F) Flow cytometry analysis in WT and GP130 KO (#2 and #7) GSC#1. Histogram plots are represented with isotype Ig (red) and GP130 staining (blue). (G) Western blot analysis of total protein lysates from WT and GP130 KO (#2 and #7) GSC#1 using the indicated antibodies. Both GAPDH and STAT3 serve as loading controls. (H) LDA were performed in WT (green) and GP130 KO (#2, black, and #7, red) GSC#1 in APLN-only containing medium. Plot is representative of two independent experiments. (I) Tumorspheres per FOV were manually, single-blindly counted in WT and GP130 KO (#2 and #7) GSC#1, cultured in MF and APLN-only medium. Data are presented as the mean ± SEM of three independent experiments. Each dot (n > 25) represents one sample count. (J) Western blot analysis of total protein lysates from WT and GP130 KO#2 cells in APLN-only containing medium (2 d) using the indicated antibodies. Both TUBULIN and STAT3 serve as loading controls. All data are representative of at least three independent experiments, unless otherwise stated. ***, P < 0.001; **, P < 0.01; *, P < 0.05 using ANOVA tests.