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. 2018 Jan 2;9(10):8941–8956. doi: 10.18632/oncotarget.23832

Figure 4. Increased level of miR-125a-5p in TIMP-1 KD clones is associated with induction of apoptosis and directly targets TIMP-1.

Figure 4

Figure 4

Figure 4

Figure 4

Figure 4

(A) Flow cytometric analysis of A549 and H460 cells treated with 0.5 μM Staurosporine for 1 hour and stained with Annexin V-PE and 7AAD. Representative figures showing more apoptosis or cell death seen in TIMP-1 KD cells. Lower Right quadrant-Annexin positive cells, showing early apoptosis; Upper Right quadrant-Annexin positive, showing late apoptosis; Upper Left quadrant showing dead cells. Only live cells were considered for analysis (P1). Graphical representation of the data (% apoptotic cells ± SEM) from two independent experiments is shown on the right. (p < 0.02*, p < 0.002**, t-test). (B) A549 cells were transfected with miR-125a-5p mimic and stained with Hoechst 33342. MiR-125a-5p cells showed significant increase in pyknotic nuclei as represented in the figure and graph below (p = 0.0008). The graph represents average value of 3 experiments ± SD. (C) A549 cells were transfected with miR-125a-5p mimic (10 nM) followed by western blot analysis to determine the level of TIMP-1 protein. The cells were harvested at 24, 48, 72, 96 and 120 hours with maximum TIMP-1 downregulation at 48 hours (top). Introduction of miR-125a-5p mimic induced downregulation of TIMP-1 after 48 hours in A549 cells. Representative figure showing upregulation of TIMP-1 with addition of miR-125a-5p antagomirs (5 nM) increased the expression of TIMP-1 level over 5 days. (D) Upregulation of p53 in A549 cells after TIMP-1 knockdown: Transfection of miR-125a-5p mimic upregulates p53 and inhibitors of miR-125a-5p downregulates p53 in A549 cells. (E) Status of pro and antiapoptotic proteins: With TIMP-1 knockdown the anti-apoptotic proteins phospho-Akt and Bcl2 levels were down-regulated and the prp-apoptotic proteins Bax and phospho-p38MAPK were upregulated. The protein expression was normalized to β-actin. Data is representative of three independent experiments. (F) Representative protein levels of mdm2: mdm2 phosphorylation is reduced after TIMP-1 knockdown, the total protein level remains unaltered.