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. 2014 Feb;34(3):533–550. doi: 10.1128/MCB.01043-13

FIG 8.

FIG 8

Concurrent knockdown of miR-200 and miR-183 cluster miRNAs increases invasion. (A) Immunoblotting from IPs performed from HCT116-p21−/− cytoplasmic extracts shows specific enrichment of Ago2 in the Ago2 IP. HC corresponds to the antibody heavy chain. (B) Enrichment of miR-183 cluster miRNAs and miR-16 (a control miRNA) in Ago2 IPs performed in duplicate from cytoplasmic extracts of HCT116 -p21+/+ and -p21−/− cells was measured by RT-qPCR of pulldown RNA. IgG IP was used as a negative control. (C) HCT116-p21−/− cells were transfected with CTL, miR-183, or miR-96 mimics for 48 h, and miR-183 and miR-96 levels were measured by RT-qPCR. (D) HCT116-p21−/− cells were transfected in duplicate with CTL or miR-183 mimics for 48 h, and enrichment of miR-183 and miR-16 in Ago2 IPs from cytoplasmic extracts was measured by RT-qPCR normalized to U6. (E) HCT116-p21−/− cells were transfected with CTL or miR-96 mimics in duplicate for 48 h, and enrichment of miR-96 in Ago2 IPs from cytoplasmic extracts was measured by RT-qPCR normalized to U6. IgG IP was used as a negative control. (F) Parental HCT116 cells were transfected in duplicate for 48 h with a control antisense oligonucleotide or a pool of antisense oligonucleotides (miR inhibitor pool) targeting miR-200a, miR-183, miR-96, and miR-182, and knockdown efficiency of miR-183 and miR-182 was assessed by RT-qPCR. (G) Parental HCT116 cells were transfected with a control antisense oligonucleotide or the miR inhibitor pool targeting miR-200a, miR-183, miR-96, and miR-182 for 48 h, and the effect on invasion was assessed by Matrigel invasion assays. (H) RT-qPCR was performed with HCT116 cells transfected with the miR inhibitor pool to determine changes in mRNA levels of EMT markers. The housekeeping gene UBC was used as a negative control. (I) Immunoblotting was performed with HCT116 cells transfected with the miR inhibitor pool to determine changes in SLUG protein levels. Error bars represent means ± standard deviations of three independent experiments. #, P < 0.01; ##, P < 0.001.