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. 2018 Dec 7;11(9):761–769. doi: 10.1093/jmcb/mjy074

Figure 1.

Figure 1

AFF3 is required for the silencing of XIST in terminally differentiated cells. (A) RNA-Seq showing the efficiency of shRNA-mediated AFF3 knockdown in HEK293T cells. (B) XIST RNA level increased by knockdown of AFF3 in HEK293T cells. (A and B) The y-axes represent RPKM values. RNA-Seq data in control and AFF3 knockdown HEK293T cells were downloaded from GSE34097. (C) RT-qPCR showing the efficiency of AFF3 knockdown by an independent shRNA in HEK293T cells. (D) RT-qPCR confirming the increase of XIST RNA level by an independent AFF3 shRNA in HEK293T cells. (E) XIST RNA FISH in control and AFF3 knockdown HEK293T cells. Scale bar, 5 μm. (F) Quantification of nuclei with XIST clouds in control and AFF3 knockdown HEK293T cells. The Chi-square test shows that the difference of cells containing three XIST clouds between control and AFF3 knockdown groups is significant. (G) RT-qPCR showing the efficiency of AFF3 knockdown in IMR-90 cells. (H) RT-qPCR showing an increase of XIST RNA level by AFF3 shRNA in IMR-90 cells. (I) XIST RNA FISH in control and AFF3 knockdown IMR-90 cells. Scale bar, 5 μm. (J) Quantification of nuclei with XIST clouds in control and AFF3 knockdown IMR-90 cells. The Chi-square test shows that the difference of cells containing two XIST clouds between control and AFF3 knockdown groups is significant. (C, D, G, and H) The expression of AFF3 and XIST was normalized to the expression of GAPDH. Results shown are technical replicates from representative biological replicates. Error bars represent standard deviations. Significant differences are marked with asterisks (t-test, *P < 0.05; **P < 0.01; ***P < 0.001).