Journal: Nucleic Acids Research

Article Title: A dual-activity topoisomerase complex promotes both transcriptional activation and repression in response to starvation

doi: 10.1093/nar/gkad086

Figure Lengend Snippet: The representative SAGs and SRGs regulated by the TOP3B–TDRD3 complex. (A, B) The mRNA levels of four SAGs and three SRGs were detected by RNA-seq. GAPDH was a negative control here. The results were from three independent replicates. The p-values were calculated by DESeq2. (C, D) RT-qPCR verified the RNA levels of the representative SAGs and ACTB gene (a control). The data from FMR1-KO cells were shown here as negative controls. The starvation-induced transcription of these four genes was not dependent on FMRP. (E) Western blot detecting the protein levels of TOP3B and ACTB in different cell lines. TOP3B-KO + WT, TOP3B-KO + Vector, TOP3B-KO + Y336F were three cell lines generated with a retrovirus vector system based on TOP3B-KO HCT116 cells. WT, ectopically-expressed wild type TOP3B; Vector, pBABE-puro vector only without transgene; Y336F, ectopically-expressed TOP3B-Y336F. The numbers in the middle are the relative protein levels measured by ImageJ. (F) RT-qPCR measuring the mRNA levels of three representative genes in the cell lines (E) with or without starvation. ***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant (n = 3, two-tailed Student's t-test). Graphs generated using P-values by ANOVA test are shown in Supplementary Figure S8.

Article Snippet: For generation of the TOP3B-KO1 HCT116 cell lines with stably-expressed TOP3B or TOP3B-Y336F protein, we used the retrovirus vector system (addgene, pBABE-puro #1764, pUMVC #8449 and pCMV-VSV-G #8454).

Techniques: RNA Sequencing Assay, Negative Control, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Generated, Two Tailed Test