Fig. 4: MTOR is involved in regulating the autophagy and apoptosis of HCT-8 cells induced by C. parvum. | Communications Biology

Fig. 4: MTOR is involved in regulating the autophagy and apoptosis of HCT-8 cells induced by C. parvum.

From: MiR-199a-3p regulates HCT-8 cell autophagy and apoptosis in response to Cryptosporidium parvum infection by targeting MTOR

Fig. 4

A MTOR mRNA expression was detected with RT–qPCR(n  =  3 biologically independent samples). B Levels of phosphorylated MTOR (p-MTOR) and total MTOR were analyzed with western blotting 12 h after HCT-8 cells were infected with C. parvum sporozoites. Densitometry was used to compare the relative levels of p-MTOR protein and total MTOR protein, and the data were normalized to GAPDH(n  =  3 biologically independent samples). C Cells were transfected with si-mTOR or si-NC for 24 h and then exposed to equal numbers of C. parvum sporozoites for 12 h. Expression levels of autophagy-related proteins LC3, P62, and Beclin 1 were determined with western blotting. Protein levels of LC3-II, P62, and Beclin 1 were determined relative to GAPDH with densitometry(n  =  3 biologically independent samples). D Cells were transfected with si-MTOR or si-NC for 24 h and then exposed to equal numbers of C. parvum sporozoites for 3 h before the medium was replaced with fresh medium. The infected cells were cultured for 12 or 24 h to evaluate the parasite burden with RT–qPCR after cell invasion(n  =  3 biologically independent samples). E Cells were cotransfected with si-MTOR or si-NC and plasmid encoding mCherry–EGFP–LC3 for 24 h, and then exposed to equal numbers of C. parvum sporozoites for 12 h(n  =  3 biologically independent samples). The autophagic flux was evaluated with confocal microscopy. Numbers of yellow and free red dots in each group cell were quantified (free red dots: autophagic lysosomes; yellow dots: autophagosome, Scale bar, 5 µm). F Cells were transfected with si-MTOR or si-NC for 24 h and then exposed to equal numbers of C. parvum sporozoites for 12 h. Cell apoptosis was then detected with flow cytometry. The apoptosis rate is the sum of O1-LR and O1-UR(n = 3 biologically independent samples). All data are presented as the means ± SD of three independent experiments. Different groups were compared with a t test (*P < 0.05, **P < 0.01, ***P < 0.001).

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