Fig. 3: NLRC5 overexpression promotes autophagy flux in NMCMs under OGD stimulation.

a NLRC5, p62, Beclin-1, and LC3-II proteins were detected by Western blot in NMCMs transfected with Ad5-Ctrl or Ad5-cTnT-NLRC5 (multiplicity of infection [MOI], 100) (n = 3 each). b Representative western blot of NLRC5, p62, Beclin-1, and LC3-II in NMCMs transfected with Ad5-Ctrl or Ad5-cTnT-NLRC5 followed by 0, 3, 6 h of OGD and corresponding quantification (n = 4 each). c NMCMs were transfected with Ad5-Ctrl or Ad5-cTnT-NLRC5 and then treated with or without bafilomycin A1 (100 nM) to evaluate autophagic flux under OGD for 6 h. Representative western blot of LC3-II and p62 and corresponding quantification (n = 3 each). d Representative immunofluorescence images demonstrating NMCMs co-transfected with Ad5-Ctrl or Ad5-cTnT-NLRC5 and an adenovirus overexpressing mCherry-EGFP-LC3 for 48 h in the presence or absence of bafilomycin A1 or OGD to evaluate LC3 puncta. Yellow dots (merged red and green) indicate autophagosomes whereas red-only dots indicate autolysosomes. Representative of n = 6 experiments. Scale bar, 20 μm. e, f Quantitative analysis of autophagosomes, autolysosomes, and ratio of autolysosomes to autophagosomes in NMCMs treated as in d from six independent experiments. g Representative transmission electron microscopy (TEM) images of autophagosomes and autolysosomes structure in NMCMs transfected with Ad5-Ctrl or Ad5-cTNT-NLRC5 under OGD. Asterisks represent autophagosomes (yellow) and autolysosomes (red). Scale bar, 500 nm. Data are presented as mean ± SD. b, c, e 2-way ANOVA with Sidak multiple comparisons test. f 1-way ANOVA with Tukey multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001. e ***P < 0.001, comparison between red dots, ^##P < 0.01, comparison between yellow dots, and ^&&&P < 0.001, comparison between total of red and yellow dots. ns indicates not significant.