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. 2015 May 22;6(22):19087–19101. doi: 10.18632/oncotarget.4236

Figure 6. CDKs affect YAP localization and the motility of breast cancer cells.

Figure 6

a. Wound-healing assay in MDA-MB-231 cells transfected with control siRNA (siCTRL) or a pool of siRNAs against CDK2, data is presented as the means of the percentage of wound closure relative to the control±SD (n > 3). b. Immunofluorescence analysis of YAP localization in MDA-MB-231 treated as in a. Data is presented as percentage of cells with nuclear YAP in each condition. c. Results of the CDK inhibitor library screening evaluating YAP localization. MDA-MB-231 cells were treated for 24 h with 1 mM of inhibitor, except for SNS-032 and DINACICLIB, which were applied at 0.1 mM. The data are presented as the means of percentage of cells with nuclear YAP±range between replicates (n = 2). See Supplementary Figure S6a for representative images. d. Immunofluorescence analysis of YAP localization in MDA-MB-157 treated with the most effective CDK inhibitors (AZD5438, JNJ7706621 or PHA793887). Data is presented as percentage of cells with nuclear YAP in each condition (n = 3). See Supplementary Figure S6a for representative images. e. Western blot of total YAP and phosphorylated YAP at Ser127 (YAP-S127) in protein lysates of MDA-MB-231 cells treated with CDK inhibitors (n = 3). f. Real time RT-PCR analysis of YAP target genes (CTGF and CYR61) in MDA-MB-231 cells treated with CDK inhibitors. GAPDH was used for normalization. The data are presented as the mean±SD (n = 3). g. and h. Wound-healing assay (g) and transwell migration assay (h) using MDA-MB-231 cells stably infected with pBABE-FLAG (FLAG) or pBABE-FLAG-YAP-5SA (FLAG-5SA) after the treatment with CDK inhibitors. See Supplementary Figure S7a and b for representative images of wound-healing and transwell respectively. Experiments presented in panel d-h were performed in cells treated with AZD5438, JNJ7706621 or PHA793887. The data are presented as the means of the percentage relative to each control (DMSO)±SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student's t-test.