Fig 2: Stem-like cell marker expression in the U87 ATCC GBM cell line and the OB1, a GBM cell line isolated from a patient directly in NS34 serum-free medium, by WB (A) and immunofluorescence (B). (A) The expression of SOX2, OCT-4A, and Nanog was quantified in both the OB1 cell line isolated from a GBM diagnosed patient directly in NS34 serum-free medium and in U87, a GBM cell line acquired by ATCC. The respective OB1 differentiated cells were isolated from OB1-SF, and the serum-free GBM cells were isolated from the U87 cell line, U87-SF, as previously described, by WB. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA). Each value represents the mean ± SEM from three independent experiments; *P < .05, **P < .01. B. Immunofluorescence staining of SOX2, OCT-4A, and Nanog as well as Nestin and GFAP was performed in both the OB1 and U87 cell lines and the respective serum-free GBM cells, OB1-SF and U87-SF. 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 3: Cells labeled with fluorojade C (green) in the CA3 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 4: The proliferation of chicken primary myoblasts (CPMs) is promoted by miR-27b-3p. (a,b) The relative mRNA expression of p21 after transfection with miR-27b-3p mimic and inhibitor in CPMs. (c,d) Cell-cycle analysis of CPMs 48 h after transfection with miR-27b-3p mimic and inhibitor in CPMs, using propidium iodide staining for DNA content. (e,f) Cell growth was measured following the transfection of miR-27b-3p mimic and inhibitor in CPMs. (g,h) The proliferation of CPMs following the transfection with miR-27b-3p mimic and inhibitor was assessed by EdU incorporation. Microscopic images were obtained by a fluorescence inverted microscope (dmi8, Leica, Germany). (i,j) Proliferation rates of CPMs following transfection with miR-27b-3p mimic and inhibitor. 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 5: The facilitation of miR-27b-3p on CPMs proliferation was achieved by its target gene MSTN. (a,b) Screening of MSTN gene interference sequence and detection of interference efficiency. (c,d) The protein expression levels of MSTN in CPMs after overexpression and interference. (e,f) The relative mRNA expression of p21 after transfection with pcDNA 3.1-MSTN and pcDNA 3.1 in CPMs. (g,h) Cell-cycle analysis of CPMs 48 h after transfection with pcDNA 3.1-MSTN and pcDNA 3.1 in CPMs, using propidium iodide staining for DNA content. (i,j) Cell growth was measured following the transfection of pcDNA 3.1-MSTN and pcDNA 3.1 in CPMs. (k,l) The proliferation of CPMs following transfection with pcDNA 3.1-MSTN and pcDNA 3.1 was assessed by EdU incorporation. Microscopic images were obtained by a fluorescence inverted microscope (dmi8, Leica, Germany). (m,n) Proliferation rates of CPMs following transfection with pcDNA 3.1-MSTN and pcDNA 3.1. 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).