Fig. 4: Effect of HLA-G on transcriptomic and cellular responses of CDi-on-chip to various stimulants.

A Immunohistochemistry of human fetal membranes, with HLA-G staining brown. B Phase-contrast images of WT and PGRMC2 KO CTC. Scale bar, 100 µm. C Western blot analysis of HLA-G expression in WT and HLA-G KO CTCs. Beta-actin serves as control. D Volcano plots of genes involved in inflammation and immunity in HLA-G KO CTC and DEC compared to WT in control conditions. Significantly downregulated genes (–log₁₀ adj. p-value > 1, log₂ fold change < –1) are shown in blue dots, while significantly upregulated genes (–log₁₀ adj. p-value > 1, log₂ fold change > 1) are shown in red dots. GSEA dot plots of top significantly-enriched KEGG pathways in (E) HLA-G KO CTC and (F) corresponding DEC. Circle size denotes number of enriched genes in a pathway. Gene ratio denotes the quotient between number of enriched genes and total number of genes in the said pathway. Color denotes p value. G Venn diagram of common differentially expressed genes in HLA-G KO CTC compared to wild-type CTC and their corresponding DEC in various conditions. GSEA ridge plots of top significantly-enriched KEGG pathways in HLA-G KO CTC and their enrichment scores (x-axis) in response to (H) LPS, (I) pIC and (J) mOS. Peak corresponds to enrichment score; enrichment score below 0 denotes suppressed pathways, while enrichment score above zero denotes activated pathways. Color denotes p-value. Measurement of (K) chorionic IL-6, IL-10, TNF-ɑ and GM-CSF, (L) decidual GM-CSF, (M) sHLA-G and (N) progesterone production in CDi-on-chip (at least n = 4 devices from one placenta per treatment group). Data are presented as mean ± SEM. Statistics: Multiple student’s t-tests (two-tailed). O Quantification of CD45⁺ cell migration from DEC into CTC (n = 5 devices from one placenta per treatment group). Data are presented as median (2nd quartile; solid line) and 1st and 3rd quartiles (dashed lines). Statistics: Multiple Mann Whitney U tests (two-tailed).