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. 2021 Feb 1;10:e61257. doi: 10.7554/eLife.61257

Figure 5. ESF-derived IL15 promotes NK and trophoblast migration in trans-well assays.

Figure 5.

(A) Cartoon of trans-well migration assay comparisons. Cells that migrated to the lower chamber were quantified using the CellTiter-Glo luminescent cell viability assay after 8 hr. (B) Primary natural killer (NK) cells. Raw luminescence data (RLU) from cells in the lower chamber is shown in the upper panel, mean difference (effect size) in experiment minus control luminescence values are shown as dots with the 95% confidence interval indicated by vertical bars in the lower panel; distribution estimated from 5000 bootstrap replicates. The mean difference between Media and media supplemented with recombinant human IL15 (Media+rhIL15) is 0.432 [95.0% CI: 0.376–0.492]; p = 0.00. The mean difference between ESFs transiently transfected with non-targeting siRNA (NT siRNA) and HAND2-specific siRNAs (siHAND2) is 0.847 [95.0% CI: 0.774–0.936]; p = 0.00. The mean difference between ESFs transiently transfected with NT siRNA and IL15-specific siRNAs (siIL15) is −0.223 [95.0% CI: −0.256 – −0.192]; p = 0.00. The mean difference between ESF media neutralized with a non-specific antibody (IgG) or IL15-specific antibody (abIL15) is −0.106 [95.0% CI: −0.147 – −0.067]; p = 0.00. n = 12. (C) Extravillous trophoblast cell line HTR-8/SVneo. Raw luminescence data (RLU) from cells in the lower chamber is shown in the upper panel, mean difference (effect size) in experiment minus control luminescence values are shown as dots with the 95% confidence interval indicated by vertical bars in the lower panel; distribution estimated from 5000 bootstrap replicates. The mean difference between Media and Media+rhIL15 is 0.482 [95.0% CI: 0.193–0.701]; p = 0.002. The mean difference between ESFs transiently transfected with NT siRNA and siHAND2 is 0.463 [95.0% CI: 0.291–0.559]; p = 0.00. The mean difference between ESFs transiently transfected with NT siRNA and siIL15 is −0.598 [95.0% CI: −0.698–0.490]; p = 0.00. The mean difference between ESF media neutralized with IgG or abIL15 is −0.267 [95.0% CI: −0.442 – −0.151]; p = 0.0004. n = 12. (D) Model of HAND2 functions in the endometrium. During the proliferative phase HAND2 inhibits IL15, and thus the migration of uNK and trophoblast cells. In the receptive phase, HAND2 activates IL15, which promotes migration of uNK and trophoblast cells. In the receptive phase and early pregnancy (EP), HAND2 suppresses estrogen signaling by down-regulating FGFs and directly binding and inhibiting the ligand-dependent transcriptional activation function of ESR1. During late pregnancy/term (LP/Term), reduced HAND2 expression mitigates its anti-estrogenic functions. Parturition signal unknown (???). ESF = endometrial stromal fibroblasts (proliferative phase), DSC = decidual stromal cells (receptive phase), LE = luminal epithelium.

Figure 5—source data 1. Raw and log transformed luminescence data for NK trans-well migration assays.
Figure 5—source data 2. Raw and log transformed luminescence data for HTR-8 trans-well migration assays.