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. 2022 Jan 20;11(3):346. doi: 10.3390/cells11030346

Figure 4.

Figure 4

Antifibrotic effect of NEN mediated by mTORC1 pathway blockade and cell cycle arrest. (A) The correlogram was derived from reanalysis of a public dataset (GEO accession #: GSE68239). Blue represents a significant positive correlation (p < 0.05), red represents a significant negative correlation (p < 0.05), and white represents a nonsignificant correlation (p > 0.05). (B–D,G,H): Blank or TGF-β1-induced A549 cells were treated with or without NEN (0.5 μM) for 24 h. The expression patterns of proteins in (B) the mTORC1 pathway, including mTOR and phosphorylated mTOR (S2448), S6 and phosphorylated S6 (S235/236), 4EBP1 and phosphorylated 4EBP1 (T70), SQSTM1/p62, Beclin-1 and LC3B (I and II), and in (G), the cell cycle pathway, including CDK2, Cyclin A, CDK4, and Cyclin D1, were detected by an immunoblotting assay; GAPDH was used as an internal reference. (C) Immunofluorescence staining was performed to detect the cellular localization of SQSTM1/p62, and nuclei were stained with DAPI. (D) The relative mRNA levels of ATGs (ATG5, ATG7, and ATG12) were detected by qPCR and normalized to the GAPDH level. (H) Cells were stained with JC-1 solution, and the cell cycle was analysed and quantified by flow cytometry. (E,F,J): DHLF-IPF cells were treated with a vehicle control or NEN (1 μM) for 24 h. Changes in the expression of proteins in (E) the PI3K/mTORC1 pathway, including Akt and phosphorylated Akt (S473), mTOR and phosphorylated mTOR (S2448), S6 and phosphorylated S6 (S235/236), 4EBP1 and phosphorylated 4EBP1 (T70), SQSTM1/p62, Beclin-1 and LC3B (I and II), and in (I), the cell cycle pathway, including CDK2, Cyclin A, CDK4, and Cyclin D1, were detected by an immunoblotting assay. GAPDH was used as an internal reference. (F) Relative ATG5 mRNA levels were detected by qPCR and normalized to the GAPDH level. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.