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 incubation (rAPLNR). Line delineates the mean, and boxes show upper and lower quartiles. Data are representative of three independent experiments. (E) Tumorspheres per FOV were manually, single-blindly counted in similarly transfected cells cultured in MF APLN-only medium. Data are presented as the mean ± SEM of three independent experiments. Each dot (n > 25) represents one sample count. All data are representative of at least three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 using ANOVA tests.