Skip to main content
. 2022 Apr 24;29(11):2137–2150. doi: 10.1038/s41418-022-01004-0

Fig. 5. CTSB inhibition induces neuronal cell apoptosis in mouse cerebrocortical cultures.

Fig. 5

A Microscopic images of propidium iodide (PI)-positive dead cells (upper) and Hoechst 33258-stained total nuclei (lower) in mouse cerebrocortical cultures 18 h after the addition of 20 µM CA-074Me in the presence or absence of 100 µM Trolox, 1 µg/ml cycloheximide (CHX), or 100 µM zVAD. CA-074Me markedly increased PI-positive dead cells and Hoechst stain revealed their condensed nuclei. Arrowheads indicate typical morphology of small, bright nuclei of apoptotic cells. The scale bar of PI images is 100 μm, and Hoechst 33258 is 25 μm. B Quantification of PI-positive dead cells (left), Hoechst 33258-stained condensed apoptotic nuclei (middle), and LDH release (right) 18 h after the addition of CA-074Me in the presence or absence of Trolox, cycloheximide, or zVAD to mouse cerebrocortical cultures (mean ± SEM, n = 3 cultures), *p < 0.05, **p < 0.01, or ***p < 0.001 by ANOVA with post-hoc Dunnett’s test. CHX and zVAD decreased nuclear pyknosis induced by CA-074Me, whereas Trolox did not affect CA-074Me-induced apoptotic neuronal cell death. C Western blot analysis for cleaved/active caspase-3 in mouse cerebrocortical cultures. Protein samples were prepared 18 h after sham wash (CTRL) or exposure to 20 μM CA-074Me in the presence or absence of Trolox, CHX, or zVAD. CA-074Me induced caspase-3 activation, which was markedly attenuated by CHX or zVAD, but not by Trolox. D Quantification of LDH release resulting from neuronal cell death 9 h after exposure to SNOC (mean ± SEM, n = 3 cultures), ***p < 0.001 by two-tailed Student’s t-test.