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. 2021 Aug 27;10(9):2214. doi: 10.3390/cells10092214

Figure 6.

Figure 6

Knockdown of p21 impairs the fusion ability of BeWo and JEG-3 cells. (A) Left panel: genome-wide profiling of BeWo cells treated with sicon or sip21 for 48 h. Total RNAs were extracted from three independent experiments. Gene expression was analyzed using HumanHT-12 v4 beadchip array. Genes with a p-value < 0.05 are included. Heatmap of the most differently expressed genes are shown (greater than 1, red color code; below 1, blue color code). Right upper panel: heatmap of fusion-related genes is depicted. Right lower panel: western blot analysis was performed with cellular lysates from BeWo cells treated with scrambled siRNA (sicon) or siRNA targeting the untranslated region (UTR) of p21 (sip21). GAPDH was taken as the loading control. (B) Gene analysis of BeWo cells depleted of p21. The mRNA levels of p21 (CDKN1A), syncytin-2 (HERV-FRD), β-hCG (CGbeta5), and GCM1 are shown. GAPDH was used as the housekeeping gene control. The results are presented as RQ with minimum and maximum range (n = 4). (C) Gene analysis of JEG-3 cells depleted of p21. The mRNA levels of p21 (CDKN1A), syncytin-2 (HERV-FRD), β-hCG (CGbeta5), and GCM1 are shown. GAPDH was used as the housekeeping gene control. The results are presented as RQ with the minimum and maximum range (n = 5). (D) BeWo cells, treated with sicon, sip21, or mixed siRNAs against the coding region of p21 (sip21 #2) for 24 h, were incubated with forskolin or DMSO for another 48 h. Treated BeWo cells were stained for the fusion marker β-hCG (red) and DNA (DAPI, blue). Examples are shown. Scale: 10 µm. (E) BeWo cells were treated with sicon, sip21, or sip21 #2. After 24 h, the syncytin-2 promoter plasmid was transfected for 48 h. Luciferase assays of BeWo cells for syncytin-2 promoter activity is shown as mean value with SD (n = 3). Dot, square, and triangle show the individual data points of sicon, sip21 and sip21 #2, respectively. (F) Western blot analysis as transfection control. GAPDH was used as the loading control. Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.