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. 2018 Oct 12;293(47):18071–18085. doi: 10.1074/jbc.RA118.004251

Figure 2.

Figure 2.

The Hippo pathway mediates the effect of cell density on COX-2 induction. A, Hippo pathway regulates the induction of COX-2 by IL-1β. H358 cells were transfected with the indicated siRNAs and treated with IL-1β for 2 h. Cells were then lysed and analyzed by immunoblotting (IB) with the indicated antibodies. B, knockdown of YAP/TAZ enhances whereas knockdown of LATS1/2 diminishes the transcription of COX-2. H358 cells transfected with control siRNA or siRNA against YAP/TAZ or LATS1/2 were treated with IL-1β for the indicated duration of time. Cells were harvested, and total RNAs were extracted for measurement of COX-2 mRNA level by qRT-PCR. n = 3 independent experiments. Data are presented as mean ± S.D. Error bars represent S.D. *, p < 0.05 (compared with siNC group at the same induction time point), by unpaired, two-tailed Student's t test. C and D, knockdown of YAP/TAZ enhances COX-2 induction by IL-1β, whereas knockdown of LATS1/2 diminishes it. H358 cells transfected with control siRNA or siRNA against YAP/TAZ or LATS1/2 were cultured onto cover glasses, treated with or without IL-1β for 2 h, and fixed for immunofluorescence staining using the indicated antibodies (C). The relative mean fluorescence intensity (anti-COX-2) of each cell was determined. n = 3 independent experiments. Data are presented as mean ± S.D. Error bars represent S.D. *, p < 0.05; and **, p < 0.01, by unpaired, two-tailed Student's t test (D). E, LATS1/2 regulate COX-2 induction through YAP and TAZ. H358 cells were transfected with siRNAs targeting LATS1/2, YAP/TAZ, or both and then treated with IL-1β for the indicated time. Cells were harvested for immunoblotting with the indicated antibodies. F, overexpression of YAP and TAZ attenuates the induction of COX-2 by IL-1β. HeLa cells were transfected with FLAG-YAP or FLAG-TAZ and treated with IL-1β for the time indicated. Cells were then lysed and analyzed by immunoblotting with the indicated antibodies. G, knockdown of YAP/TAZ abolishes the effect of cell density on COX-2 induction by IL-1β. H358 cells plated at low or high cell density were transfected with control siRNA or siRNAs against YAP and TAZ for 48 h and then stimulated with IL-1β for the time indicated. Cells were then lysed and analyzed by immunoblotting with the indicated antibodies. H, overexpression of YAP(5SA) abolishes the effect of cell density on COX-2 induction by IL-1β. H358 cells plated at low or high cell density were transfected with empty vector (EV) or a plasmid expressing the constitutively active YAP mutant YAP(5SA) for 24 h and then stimulated with IL-1β for the indicated duration of time. Cells were then lysed and analyzed by immunoblotting with the indicated antibodies. NC, negative control.