Journal: iScience
Article Title: The microcephaly-associated protein YIPF5 differentially regulates ER export
doi: 10.1016/j.isci.2026.114791
Figure Lengend Snippet: YIPF5 regulates cellular and neuronal migration in vitro and in vivo (A) MCF10A WT, YIPF5-KO, and YIPF5-KO cells re-expressing either the I98S YIPF5 mutant or WT YIPF5 at low (blue) or high (violet) levels were plated in Incucyte Imagelock 96-well plates and a wound healing assay performed using the Incucyte Wound Maker 96-Tool on the IncuCyte system. Displayed is the relative wound density at different time points as mean values ± SEM from three biological replicates. Statistical significance was evaluated using a two-way ANOVA with Tukey’s multiple comparisons test, with ∗∗ denoting p < 0.01 and ∗ indicating p < 0.05. (B) Schematic representation illustrating the in utero electroporation procedure at embryonic day 13.5 (E13.5) with a GFP-labeled plasmid encoding either shLuciferase or shYIPF5, followed by brain removal at E15.5. (C) Scheme of a coronal section of a mouse embryonic brain schematizing the position of the electrodes, the ventricles and the developing neocortex with the classification into distinct zones, ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), and cortical plate (CP). This classification traces the cortical migration pattern of GFP + electroporated cells. Illustrations created with BioRender.com using an IHC image deriving from our murine brain. (D) Murine brains electroporated at E13.5 with plasmids expressing control shLuciferase or shYIPF5 were harvested at E15.5, fixed, sectioned with a vibratome, and immunostained for GFP to visualize transfected cells. Nuclear staining with Hoechst (not shown) facilitated the differentiation of brain regions, including VZ, SVZ, IZ, and CP. Representative images are shown. (E) Quantification of the proportion of EGFP + cells within specified cortical regions. Displayed are the means ± SEM (shLuciferase, n = 5 brains, 225 EGFP + cells; shYIPF5, n = 5 brains, 257 EGFP + cells). Statistical analysis: multiple unpaired t test. (F) Morphometric analysis of CP-localized neurons of shLuciferase and shYIPF5 electroporated brains. Images are maximum projections of sequential z sections, the inverted EGFP signal is depicted in black. (G) Quantification of neuron morphologies from shLuciferase ( n = 6 brains, 6 sections, 93 EGFP + cells) and shYIPF5 ( n = 5 brains, 6 sections, 71 cells) groups. Neurons were categorized into two groups: unbranched uni/bipolar and exhibiting complex arborizations. Mean values with standard deviation are shown, statistical analysis: one-way ANOVA with Šídák’s multiple comparisons test, ∗ p < 0.05. (H) Leading process orientation analysis of CP-localized neurons from brain sections as shown in (F). Arrowheads indicate examples of tilted neurites. The angles between the leading neurites of EGFP + neurons and the CP surface were calculated using the measurement feature in ImageJ. The frequency of angle values was categorized into specified ranges, expressed as percentages of the total number of values. The sample sizes comprised 92 EGFP + cells from six sections from six brains for the shLuciferase group and 96 EGFP + cells from nine sections from five brains for the shYIPF5 group. Displayed is the mean ± SEM. Independent unpaired t tests were conducted for the three angle ranges, with a significance level set at ∗ p < 0.05.
Article Snippet: For live imaging, cells were cultured in a 96-Well Cell Imaging Plate with a cover glass bottom (Eppendorf, Cat. No. 0030 741.030).
Techniques: Migration, In Vitro, In Vivo, Expressing, Mutagenesis, Wound Healing Assay, In Utero, Electroporation, Labeling, Plasmid Preparation, Control, Transfection, Staining, Standard Deviation
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