Journal: International journal of molecular sciences

Article Title: Combination of Epigallocatechin-3-Gallate and Tramiprosate Prevent Accumulation of Intracellular Aβ and Dysfunctional Autophagy-Lysosomal Pathway at Earliest Stage of Transdifferentiation of Mesenchymal Stromal Cells into PSEN1 E280A Cholinergic-like Neurons.

doi: 10.3390/ijms26083756

Figure Lengend Snippet: Figure 3. Flow cytometry and immunofluorescence analysis of proliferation marker Ki67 and cluster of differentiation 73 (CD73) during the process of transdifferentiation of menstrual mesenchymal stromal cells (MenSCs) into cholinergic-like neurons (ChLNs). Representative histograms showing Ki67 (A–E) and CD73 (F–J) flow cytometry analysis performed in wild-type (WT) PSEN1 (blue his- tograms) and E280A (red histograms) MenSCs after 0 (A,F), 1 (B,G), 3 (C,H), 5 (D,I), and 7 (E,J) days of culture in Ch-N-Rm. (K,L) Bar charts represent the quantitative analysis of the positive population. Representative images showing Ki67 (green) and CD73 (red) double-stained WT PSEN1 (M-Q) and E280A (R–V) MenSCs after 0 (M,R), 1 (N,S), 3 (O,T), 5 (P,U), and 7 (Q,V) days of culture in Ch-N-Rm. Nuclei were stained with Hoechst dye (blue; M–V). (W,X) Bar charts represent the mean fluorescent intensity (MFI) quantification of images obtained by immunofluorescence analysis. The histograms and images represent one out of three independent experiments. Significant values were determined by two-way ANOVA with a Tukey post hoc test; ** p < 0.01, *** p < 0.001 intergroup comparison; n.s. = not significant. Image magnification 20×.

Article Snippet: For the analysis of differentiation, neuronal, Alzheimer’s disease, oxidative stress, autophagy, and cell death-related markers, cells treated under different conditions were washed and incubated with 10% bovine serum albumin (BSA) blocking solution for 30 min. For the analysis of neuronal Alzheimer’s disease, oxidative stress, autophagy, and cell death-related markers, cells were incubated overnight with primary antibodies against Ki-67 (rabbit; abcam; cat. #ab15580, Cambridge, UK), CD73 (mouse; Elabscience; cat. #E-AB-F1242E, Houston, TX, USA), NeuN (rabbit; abcam; cat. #ab177487, Cambridge, UK), ChAT (ChAT, Millipore; cat. #AB144 P, Burlington, MA, USA), protein amyloid β1-42 (mouse, Millipore; cat. #MABN639, Burlington, MA, USA), oxidized DJ-1 (oxDJ-1; spanning residue C106 of human PARK7/DJ1; oxidized to cysteine sulfonic acid (SO3); rabbit, abcam; cat. #ab169520, Cambridge, UK), and LC3-II (rabbit, Novusbio; cat. #NB100-2220, Centennial, Colorado, USA) or 1X CellEvent Caspase3/7 Detection Reagent (Invitrogen, cat. # C10423, Carlsbad, CA, USA).

Techniques: Flow Cytometry, Immunofluorescence, Marker, Staining, Comparison