Fig 2: Cells labeled with fluorojade C (green) in the CA1 hippocampal region of (A–C) senescence-accelerated mice resistant (SAMR)-1, (D–F) Senescence Accelerated Mouse-Prone 8 (SAMP-8), (G–I) Li, (J–L) EE, and (M–O) Li + EE. Cellular nuclei were labeled with DAPI (blue). Images were obtained using an inverted DMi8 (Leica, Wetzlar, Germany) microscope with a 10× objective. Scale bar: 100 µm.

Fig 3: The inhibition of miR-27b-3p on MSTN is different between CPMs proliferation and differentiation. (a) The relative expression of MSTN during CPMs’ proliferation and differentiation (GM 50% and GM 100% represent the proliferation density of 50% and 100%; DM1-DM 4 represents differentiation from one to four days. (b,c) The mRNA expression levels of differentiation marker genes after transfection with pcDNA 3.1-MSTN and siR-MSTN in CPMs. (d,e) The protein expression levels of MyHC after transfection with pcDNA 3.1-MSTN and siR-MSTN in CPMs. (f,g) MyHC staining of CPMs at 72 h after transfection with pcDNA 3.1-MSTN and siR-MSTN in CPMs. Microscopic images were obtained by a fluorescence inverted microscope (dmi8, Leica, Germany). (h,i) Myotube area (%) of CPMs 72 h after overexpression and inhibition of MSTN. (j,k) The proliferating (GM) and differentiating (DM) CPMs were transfected with miR-27b-3p mimic, respectively, and the relative MSTN mRNA expression levels were then analyzed. In all graphs, the results are shown as mean ± SEM (standard error of the mean) (n = 3). Statistical significance of differences between means was assessed, using the unpaired Student’s t-test (* p < 0.05; ** p < 0.01) vs. NC (negative control).

Fig 4: The GSC maintenance (A) and clonogenic Assay (B). (A) The human tumor cell lines were established in our laboratory. The use of patients' surgical specimens for the establishment of cell lines for in vitro and in vivo studies had written informed consent from the patients and was approved by the Brazilian Ministry of Health Ethics Committee under Institutional Review Board (Research Ethics Committee of Hospital Universitário Clementino Fraga Filho) consent, CEP-HUCFF no. 002/01. Cells were grown and maintained in DMEM-F12 supplemented with 10% FBS. Culture flasks were maintained at 37°C in a humidified 5% CO2 and 95% air atmosphere. GSCs were maintained as tumor-sphere cultures in NeuroBasal medium supplemented with sodium pyruvate, glutamine, B27 supplement, EGF, basic FGF, penicillin, and streptomycin. The OB1 differentiated cell line was obtained by the removal of EGF and bFGF and the addition of 10% fetal bovine serum. (B) To estimate stem cell frequency in the cell population, tumor-sphere formation was assayed with 2 cells per well in in 96-well plates in NeuroBasal medium supplemented with sodium pyruvate, glutamine, B27 supplement, EGF, basic FGF, penicillin, and streptomycin. During 4 weeks of culture, sphere-positive wells were scored by observation under an inverted microscope with phase-contrast optics. The image is representative of three independent experiments. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).

Fig 5: Depletion of retromer or retromer-associated SNXs decreases EGFR protein levels. (A) Steady-state HeLa control, Vps35 KO, SNX1/2 dKO, SNX3 KO, and SNX27 KO cell monolayers grown in complete-DMEM media on coverslips were fixed in ice-cold methanol, stained with EGFR antibody, and count-stained with DAPI. Images were captured on a Leica SP8 DMi8 confocal microscope using a 60× glycerol objective (Scale Bar, 10 µm). (B) Equal amounts of protein samples from HeLa control, Vps35 KO, SNX1/2 dKO, SNX3 KO, and SNX27 KO cells were subjected to SDS-PAGE and immunoblotting with the antibodies against EGFR, SNX1, SNX2, SNX3, SNX27, and Vps35, whereas tubulin served as a loading control. Representative blots from at least three independent experiments are shown. (C) The fold differences for EGFR protein levels are presented (means ± SEM). Two-tailed student’s t-test indicated the difference between HeLa control and HeLa KO cells, ** p < 0.01, HeLa control vs. Vps35 KO; *** p < 0.001, HeLa control vs. SNX1/2 dKO; *** p < 0.001, HeLa control vs. SNX3 KO; *** p < 0.001, HeLa control vs. SNX27 KO.