Fig 2: qRT-PCR validation of a subset of PAS-associated DE genes and extending analysis of B2M overexpression in PAS to the protein level. (A) We used qRT-PCR to validate expression, at the mRNA level, of specific genes identified as DE in the microarray analysis. For these experiments, we assayed two of the original PAS samples and five that were subsequently collected (n = 7). An equal number of CTB samples isolated from nPTB cases served as controls. The results confirmed higher expression of DOCK4, B2M, PAPD4, and ANKRD44 in accreta vs. nPTB; PRL was down-regulated. Circles represent nPTB samples and triangles represent PAS samples. Asterisks indicate significant differences between nPTB and PAS samples within each experiment (P < 0.05). (B) To confirm that differences in mRNA expression were also observed at the protein level, we immunolocalized B2M in biopsies from nPTB (n = 5) and PAS cases (n = 7). Lower immunoreactivity with anti-B2M was observed in association with CK-positive CTBs from nPTB (A and B) as compared to a PAS (increta; ICT) case (C and D). (C and D) Immunoblot analyses of CTB lysates corroborated these observations (n = 3 technical replicates). On average, B2M was expressed at fivefold higher levels by CTBs isolated from PAS vs. nPTB cases (P < 0.01). dd, ??; FV, floating villi; STBs, syncytiotrophoblasts; ACT accreta; PCT, percreta. (Scale bars, 25 µm.)

Fig 3: Overexpression of DOCK4-increased CTB invasion. Cells for immunoblotting or quantification of invasion were processed at the conclusion of the experiment (36 h). (A and B) Immunoblotting and scanning densitometry showed significantly higher DOCK4 expression (P < 0.01) associated with the CTBs that were transduced with the DOCK4-containing vector as compared to control cells that received the empty vector (n = 3 biological replicates). (C) Up-regulation of DOCK4 increased CTB invasion, on average, by ~3.7-fold (double cross; ANOVA; P < 0.001) as compared to the level observed in the control cultures (n = 3 biological replicates). Asterisks and double dagger symbols indicate significant differences between DOCK4+ and control within each experiment (P < 0.01).

Fig 4: USP36 regulates the ubiquitination level of DOCK4. (A) String website was performed to predict that USP36 can bind to DOCK4. (B) Immunoprecipitation analysis was performed with cell lysates of TECs with anti-USP36 or anti-DOCK4 antibodies, n = 3. (C) Interaction of USP36 and DOCK4 in TECs visualized by Duolink proximity ligation assay, n = 3. (D) RT-qPCR analysis of the mRNA level of DOCK4 after knockdown or overexpression of USP36. Unpaired, two-tailed t tests were used to compare groups, n = 3, ns, p > 0.05. (E,F) WB analysis of the protein levels of USP36 and DOCK4 in TECs after knockdown or overexpression of USP36. Data are presented as the mean ± S.D, n = 3, *p < 0.05, **p < 0.01. (G,H) WB analysis of the protein levels of USP36 and the ub-DOCK4 in TECs after knockdown or overexpression of USP36. Data are presented as the mean ± S.D, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. (I) Ub-DOCK4 was detected by Duolink proximity ligation assay after knockdown or overexpression of USP36. Scale bars = 25 µm, n = 3. (J) Duolink proximity ligation assay analysis of ub-DOCK4 expression after knockdown or overexpression of USP36. Data are presented as the mean ± S.D, n = 3, **p < 0.01, ***p < 0.001.

Fig 5: Validation of CTB overexpression of DOCK4 in PAS versus nPTB cases. (A) We confirmed up-regulation of DOCK4 at the protein level. Immunolocalization failed to detect binding of anti-DOCK4 to STBs or CTBs in floating villi (FV) from either controls or cases (A and C). Analysis of basal plate biopsies from pregnancies complicated by nPTB (n = 5) revealed a relatively faint but consistent signal that was associated with invasive CTBs (B), which was significantly stronger in equivalent PAS samples (D, n = 7). (B and C) Immunoblotting confirmed and enabled quantification of this result. Four of the six CTB lysates isolated from PAS cases had an immunoreactive band that corresponded to the molecular weight of DOCK4 (225 kDa) and lower molecular weight splice variants. Notably, these bands were not observed in the two accreta samples. DOCK4 expression was ~threefold higher in the setting of PAS as compared to PTB. ICT, increta; ACT accreta; PCT, percreta; IB, immunoblot. (Scale bars, 25 µm.)