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. 2014 Nov 13;4:7026. doi: 10.1038/srep07026

Figure 2. Increased apoptosis in Ubb−/− cells during culture in vitro.

Figure 2

(a) Cells isolated from wild-type (Ubb+/+) and Ubb−/− embryonic brains (n = 3 each) on 14.5 dpc were cultured in vitro for 3 days. To determine the percentages of cells undergoing apoptosis, the total number of TUNEL+ cells was divided by the total number of DAPI+ cells in three randomly selected fields for each sample. For each sample, more than 100 DAPI+ cells were counted. (b) Cells isolated from wild-type (Ubb+/+) and Ubb−/− embryonic brains on 14.5 dpc and cultured in vitro for 3 days (DIV3) were subjected to a double-labeling fluorescence TUNEL assay with the NSC marker NES, neuronal marker NeuN, and glial cell marker GFAP, and DNA was visualized with DAPI. Arrowheads indicate TUNEL+ apoptotic cells that are also positive for cell-type specific markers. (c) The percentage of apoptotic (TUNEL+) or non-apoptotic (TUNEL−) NES+, NeuN+, or GFAP+ Ubb−/− cells (n = 3) was determined in a similar manner as described in (a). (d) Immunoblot detection of apoptotic marker caspase-3 (CASP3) in wild-type (+/+) and Ubb−/− (−/−) embryonic brain lysates on 14.5 dpc (14.5 dpc) or in cells isolated from embryonic brains on 14.5 dpc and cultured in vitro for 7 days (DIV7). CASP3-immunoreactive bands were detected at 35 and 17 kDa, representing pro-caspase-3 (pro-CASP3) and active caspase-3 (active CASP3). β-Actin (β-Act) was used as a loading control. (e) Wild-type (Ubb+/+) and Ubb−/− cells on DIV13 were stained with markers for GFAP or NeuN, and DNA was visualized with DAPI. (f) On DIV3 and DIV13, the percentages of wild-type (Ubb+/+) and Ubb−/− cells (n = 3 each) positive for NeuN or GFAP were determined in a similar manner as described in (a). Representative images or immunoblot results of cells from three different embryonic brains per genotype are shown (b, d, e), and data are expressed as the means ± SEM from the indicated number of samples (a, c, e). *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ubb+/+ on each day or as indicated by bars. Scale bars, 50 μm.