Fig. 5
From: YTHDF1 links hypoxia adaptation and non-small cell lung cancer progression
The Keap1-Nrf2-AKR1C1 axis is the mediator for YTHDF1. a After DDP (15 μM) treatment in A549 cells, indicated cell extracts were examined by western blot. Antibodies: Keap1, Nrf2, AKR1C1, β-actin, Lamin B (Nuclear fraction), GAPDH (cytosol fraction). b Representative images indicating Nrf2 nuclear accumulation after YTHDF1 knockdown in A549 cells treated by 15 μM DDP. Scale bar: 10 μm. c, Representative IHC stain,ng of KP and KPY tumors for Keap1, Nrf2 and AKR1C1 expressions. Scale bar; 50 μm. d, e Effect of DDP and/or AKR1C1 inhibitor BPS on cell viabilities of indicated A549 cell lines (d), which were further validated by western blot (e). DDP: 15 μM, BPS: 40μM. Indicated cells were pretreated by DDP for 12 h followed by BPS treatment. Indicated total extracts were probed with indicated antibodies: PARP, CC3, Bcl-2, BAX and β-actin. Red stars: DDP treatment group comparison; Green stars: DDP + BPS treatment group comparison. f YTHDF1 knockdown exhibited antioxidant functions. Indicated A549 cell lines were treated with indicated doses of H2O2 for 12 h, intracellular ROS levels were measured. Representative histograms of ROS analysis are shown. g, j YTHDF1 negatively correlates with AKR1C1 expressions in NSCLC (SCC and ADC) tissues, validated by both IHC staining in TMA (g) and fresh clinical tissues using western blot (h). (i) is the quantification data for (g). (j) is the quantification data for (h). k, l YTHDF1 knockdown dependent resistance to hypoxia-induced cellular apoptosis is reversed by treating cells with 40 μM BPS. After 72 h treatment under hypoxia condition, indicated cells are stained with Annexin V/PI, and the percentage of apoptotic cells was assessed by flow cytometry (k). (l) is the quantification data for (k). Means ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; t-test
