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. 2021 Nov 15;11(11):5680–5700.

Figure 7.

Figure 7

AS1411 mislocalizes TBX3 and nucleolin to the cytoplasm and causes upregulation of TBX3 and nucleolin tumor suppressor target genes. A. Confocal microscopy images of SW1353 cells treated with 10 μM AS1411 or CRO for 48 h and processed for immunofluorescence with antibodies to TBX3 (Cy3; red channel) and nucleolin (Alexa 488; green channel). Confocal microscopic images from 20 fields of view were captured and representative images taken at 400× magnification are shown (scale bars, 20 μm). Nuclei were visualized by co-staining cells with DAPI. B. SW1353 cells were treated with 10 μM AS1411 or CRO for 48 h. Nuclear and cytoplasmic proteins were separated and analyzed by western blotting using primary antibodies to TBX3, nucleolin, GAPDH (cytoplasmic fraction marker) and p84 (nuclear fraction marker). C. Venn diagram analysis across two groups of genes showing downregulated genes in TCGA sarcoma patients expressing high TBX3 (n = 329) versus high nucleolin (NCL) (n = 189). 91 overlapping genes (hypergeometric P-value = 3.12×10-114). D. Analysis of CDKN1A and CDKN2A expression in TCGA sarcoma patient samples expressing low TBX3 and NCL versus high TBX3 and NCL. E. qRT-PCR analysis of TBX3 tumor suppressor target genes (CDKN1A (p21CIP1) and CDKN2A (p14ARF)) after 24 h and 48 h treatment with 10 μM AS1411. Student’s t-test was used to compare between groups, *P<0.05; error bars represent mean ± SEM.