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. 2018 Sep 5;22(11):5378–5393. doi: 10.1111/jcmm.13810

Figure 2.

Figure 2

Silencing of ABCB1 reduced HTR8/SV neo cell proliferation, invasion and induced cell fusion: A, Bar graph representing the relative fold change from the NC‐1 control in average ABCB1/C2 and G2 mRNA levels assessed by qPCR. ABCB1 is down‐regulated by 62% (P = 0.004), while levels of ABCC2 and ABCG2 remain stable. Levels are normalized over the geometric mean of YWHAZ and TOP1 housekeeping genes. B, Representative immunoblot of P‐gp levels in NC‐1 and Dsi2 ABCB1‐silenced HTR‐8 cells. Graph shows the relative band intensity for 170 kDa P‐gp over the housekeeping protein 14,3,3 zeta (P = 0.0012). C, Line graph showing the 24‐h growth curves of HTR8/SV neo cells following ABCB1 knockdown showing a 24% reduction in the proliferative rate in ‐Dsi2 ABCB1 ‐ versus NC‐1 control‐treated cells (P = 0.012). D, Bar graph showing the change in EVT and fusion marker mRNA levels 24 h after silencing of ABCB1 as determined by qPCR. Only fusion markers GJA1 and ERVW‐1 were increased on treatment with DSi2 ABCB1 (*P < 0.05). Data are expressed as the relative fold change from NC‐1 controls shown by the dashed line. Data were normalized as in A. E and F, Bar graphs representing the HTR8/SV neo invasion and fusion indices following transfection of NC‐1 control or Dsi2 ABCB1. Silencing of ABCB1 results in a > 90% reduction in invasive cells but stimulates a significant twofold increase in cell‐cell fusion (P < 0.0001). Representative photomicrographs in E show invading cells (located on the bottom of the 8 μm cell inserts) following H&E staining. Photographs in F show a decrease in E‐cadherin staining in ABCB1 silenced cells and an increase in nuclear size and endomitotic nuclei (*). All experiments were conducted in triplicate on three different passages of HTR8/SV neo, and all assessments were performed in duplicate, minimum. Error bars shown in all graphs are the standard deviations from the mean, and data were analysed using one‐way ANOVA and Bonferroni post hoc testing or the paired Student's t test in D. NC‐1 = universal normal control siRNA duplex. Dsi2 ABCB1 = ABCB1‐specific siRNA duplex