Journal: Cell Death & Disease

Article Title: Combined ketone body and glutamine supplementation restores aerobic energy production in AGC1-deficient neuronal progenitors

doi: 10.1038/s41419-025-08314-4

Figure Lengend Snippet: A Characterization of control and patient NPs by Immunofluorescence. NPs were cultured on matrigel-coated 24 mm coverslips and fixed with 4% PFA. Cells were then permeabilized with PBS-0.1% Triton X-100 and subjected to immunostaining with doublecortin (DCX, marker of neural stem cells, NSCs, committed to become neurons), Nestin (marker of NSCs), Musashi 1 (marker of neural precursor cells) and Synapsin 1 (marker of differentiated neurons) antibodies and nuclear staining with DAPI (DCX panels). Scale bars: 10 μm. B Representative bright-field images and quantification of total cell area of control (C1, C2, C3; gray bars) and patient NPs (P1, P2A, P2B; white bars). For each experiment, the areas of at least 10 cells were measured using NIS Elements AR software (Fiji, NIH, USA) in each of three randomly selected fields acquired with a 10× objective on an Eclipse TS100 microscope (Nikon, Japan). Data represent the means ± SD of three independent acquisitions. C Western blot (WB) analysis of 100 μg of protein of control (C1, C2, C3; gray bars) and patient NPs (P1, P2A, P2B; white bars), probed with antibodies against AGC1, AGC2, and GAPDH. Histogram graphs show the means ± SD of the densitometric ratios of AGC1 or AGC2 to GAPDH from three independent experiments. D – G Control (C1, C2 and C3, gray bars) and patient NPs (P1, P2A, P2B; white bars) were seeded at a density of 20.000 cells/cm 2 and grown for 24 and 48 h in DM, with or without 2 mM glutamine. Histograms show the means ± SD of the number of viable ( D ) and nonviable Trypan blue-stained cells ( E ) from three independent experiments. NPs were incubated with 1 μg/mL DAPI and immunostained for the proliferation markers BrdU ( F ) and Ki67 ( G ). Confocal images were acquired using a 60× objective, and the means ± SD of the percentage of BrdU + or Ki67 + / total cells were calculated from three independent experiments. H , I Activation of apoptosis in AGC1 deficient NPs. Representative WB showing the total and cleaved forms of Caspase 3 ( H ) and PARP1 ( I ) in 100 μg of control and patient NPs probed with antibodies against Caspase 3, PARP1 and citrate synthase CS. ROS production was measured in control (C1, C2, C3; gray bars) and patient NPs (P1, P2A and P2B, white bars) incubated 8 h in DM ± 2 mM glutamine and loaded with 20 μM CM-H2DCFDA (DCF, J ) or with 5 μM MitoSOXred ( K ). DCF fluorescence was measured using cytofluorimetry, while MitoSOXred fluorescence intensity was quantified via fluorescence microscopy in at least 30 cells in each experiment. Data represent the means ± SD of three independent experiments. The images shown are representative of MitoSOXred fluorescence from control C1 and patient P1 NPs incubated in DM without glutamine. Scale bars: 10 μm. *p < 0.05, **p < 0.01 ***p < 0.001 by one-way ANOVA with Tukey’s comparison test.

Article Snippet: Cells were then permeabilized with PBS-0.1% Triton X-100 and subjected to immunostaining with doublecortin (DCX, marker of neural stem cells, NSCs, committed to become neurons), Nestin (marker of NSCs), Musashi 1 (marker of neural precursor cells) and Synapsin 1 (marker of differentiated neurons) antibodies and nuclear staining with DAPI (DCX panels).

Techniques: Control, Immunofluorescence, Cell Culture, Immunostaining, Marker, Staining, Software, Microscopy, Western Blot, Incubation, Activation Assay, Fluorescence, Comparison