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. 2018 Nov 15;28(6):1023–1037. doi: 10.1093/hmg/ddy396

Figure 6.

Figure 6

Effects of the Asn492Ile PRLR variant and His188Arg mutant on proliferation, assessed by BrdU incorporation, and on apoptosis, assessed by Caspase-3/7 assays. (A) Representative images showing BrdU-immunostaining in HEK293 cells that were transfected with WT, variant Asn492Ile or mutant His188Arg PRLR. Following transfection, the HEK293 cells were exposed to 200 ng/mL PRL, and at 96 h incubated with BrdU for 15 min to assess the number of proliferating cells. Bar indicates 20 μm. (B) Quantification of the number of BrdU-immunostained cells. Cells transfected with the Asn492Ile PRLR variant showed significantly increased BrdU immunostaining, when compared to cells transfected with WT PRLR or the mutant His188Arg PRLR, thereby confirming that the Asn492Ile PRLR variant is associated with increased proliferation (Fig. 5E). Mean ± SEM from N = 5 coverslips per construct, with four images taken per coverslip. Coverslips were prepared from independent transfections that were performed on two separate days. ****P < 0.0001. (C) Quantification of Caspase-3/7-mediated apoptosis activity in transfected HEK293 cells. Cells were transfected with WT, variant Asn492Ile or mutant His188Arg, treated with 0 ng/mL PRL or 200 ng/mL PRL and assessed for apoptosis at 96 h. Data were normalized to the number of proliferating cells at hour 0. Following treatment with PRL, cells expressing the His188Arg PRLR mutant had significantly more apoptosis than WT cells or cells expressing the Asn492Ile variant. Mean ± SEM for four apoptosis assays with each assay performed with four technical replicates. **P < 0.01.