Table 1.
Primers used for quantitative gene expression analysis.
Table 2.
Antibodies used for western blotting and immunostaining.
Fig 1.
Visualization of Prl and Cts loci in TGCs.
(A) Scale diagram of the Prl and Cts gene regions on chromosome 13. BAC clones to detect Prl and Cts loci are shown by green and red lines, respectively. (B) Arrangement of Prl and Cts loci in 2C TSC and 4C, 8C, 16C, and 32C TGCs. Bars mean 5 μm. (C) Relationship between the numbers of Prl and Cts signals in a TGC nucleus. (D) Relationship between nuclear volume and numbers of Prl and Cts signals.
Fig 2.
Nuclear structures of TSCs and TGCs.
(A) Electron micrographs of TSC (Day 0) and TGC (Day 9) nuclei. (B) Localization of H3K9me2 in TSC (Day 0) (n = 341) and TGC (Day 9) (n = 226). Bars mean 20 μm.
Fig 3.
Localization of LMNB1 during TGC formation.
(A) Percentages of TGCs at days 0–9 after differentiation. The percentage of TGCs dramatically increased from days 6 to 9. (B) Western blot analysis of LMNB1 at days 0–9 after differentiation. The amount of LMNB1 protein peaked at day 3 and then gradually decreased. The data are represented as means ± SE (n = 5). Significance was determined by the t-test as a comparison between day0 and each day after differentiation. *Indicates P < 0.05, **indicates P < 0.01. (C) Localization of LMNB1 at days 0–9 after differentiation. Although almost all cells were LMNB1-high at day 0, 50% and 20% were LMNB1-low and -depleted, respectively, at day 9 (n >150, each stage). (D) The size of day 9 TGCs nucleus: LMNB1 high (n = 35), low (n = 63) and depleted (n = 27). Box plots show the central 75% (boxes), median (lines in boxes), average (x marks), and range (whiskers). (E) Localization of LMNB1 and H3K9me2 at day 9 after differentiation. White arrowheads indicate LMNB1-high TGCs and red arrowheads indicate LMNB1-low TGCs. The percentage of TGCs with peripheral H3K9me2 decreased significantly in LMNB1-low and -depleted cells. Statistical analysis was performed by the χ2 test: LMNB1 high (n = 81), low (n = 87), and depleted (n = 58). Different letters indicate significant difference (P<0.05).
Fig 4.
Knockdown of Lmnb1 in TGCs by siRNA.
siRNA-transfected TSCs were cultured for 3 days in TS medium without Fgf4. LMNB1 protein was then detected by western blotting (A), and the localization of LMNB1 and H3K9me2 was detected by immunostaining. (C) Expression levels of undifferentiated gene (Cdx2), trophoblast giant cell markers (Prl3d1, Prl2c2, and Prl4a1), labyrinth markers (Gcm1 and Syna), and spongiotrophoblast markers (Ascl2 and Tpbpa) were examined by qRT-PCR. Expression levels were normalized with Gapdh. The data are presented as means ± SE (n = 3). Significance was determined by the t-test. *Indicates P < 0.05 compared to scramble RNA.
Fig 5.
LMNB1 expression in placenta at 14.5, 16.5, and 19.5 dpc.
(A) White arrowheads indicate LMNB1-depleted TGCs. LMNB1-depleted TGCs were detected at 16.5 and 19.5 dpc. (B) Percentage of LMNB1-depleted TGCs. Statistical analysis was performed by theχ2 test:16.5 dpc (n = 105) and 19.5 dpc (n = 128). *, P < 0.05.
Fig 6.
(A) Cell viability was detected with the Live/Dead Cell Staining Kit Ⅱ. Live cells were stained by Calcein AM (green) and dead cells were stained by ethidium homodimer Ⅲ (red). The percentage of dead TGCs was significantly higher at day 9 than at day 6. (B) Effect of P4 on the viability of TGCs. The percentages of dead TGCs decreased significantly in a P4-dose-dependent manner. White circles indicate dead TGC nucleus. The data are presented as means ± SE (n = 3). The percentage of dead TGCs were analyzed by ANOVA followed by Dunnett’s test. Dunnett’s test was performed as a comparison between TSM + EtOH and each concentration of P4. Statistical significance was defined as follows: *, P < 0.05.
Fig 7.
Effect of P4 on localization of LMNB1 in TGCs.
(A) Localization of LMNB1. Bars mean 20 μm. (B) Percentages of LMNB1-high TGCs increased significantly in a P4-dose-dependent manner. Statistical analysis was performed with the χ2 test: +EtOH (n = 161), 10-8M (n = 231), 10-7M (n = 171) and 10-6M (n = 133). Different letters indicate significant difference (P<0.05).