Journal: Cell reports

Article Title: Perturbations in 3D genome organization can promote acquired drug resistance.

doi: 10.1016/j.celrep.2023.113124

Figure Lengend Snippet: Figure 1. Transcriptional activation of ABCB1 drives Taxol resistance in RPE-TxR (A) Crystal violet staining of viability assay on Taxol-naı¨ve RPE-TxS and resistant RPE-TxR cell lines. (B) Relative survival plots of the RPE-TxS and RPE-TxR cell lines. Error bars show the average ± SD of three independent experiments (n = 3) and the calculated IC50. The curve was drawn from the log(inhibitor) vs. response equation Y = Bottom + (Top Bottom)/(1 + 10^(X-LogIC50)). (C) ABCB1 mRNA levels determined by qRT-PCR and normalized to GAPDH expression levels. Error bars show the average ± SD of three independent ex- periments (n = 3). The y axis is shown in logarithmic scale. (D) Representative smRNA-FISH images of RPE-TxS and RPE-TxR for the ABCB1 gene and DAPI. The images are projections of 0.5-mm sections and a total 5 mm in thickness. Scale bar, 15 mm. (E) Quantification of the number of ABCB1 transcription sites (TS) found per cell. Error bars show the average ± SD of three independent experiments (n = 3), 60 cells per replicate and condition.

Article Snippet: Strand-specific libraries were generated using the TruSeq PolyA Stranded mRNA sample preparation kit (Illumina).

Techniques: Activation Assay, Staining, Viability Assay, Quantitative RT-PCR, Expressing

Journal: Cell reports

Article Title: Perturbations in 3D genome organization can promote acquired drug resistance.

doi: 10.1016/j.celrep.2023.113124

Figure Lengend Snippet: Figure 5. Transition to Taxol resistance is not primarily driven by repressive chromatin modifications of ABCB1 genomic locus (A) Western blot showing the levels of the chromatin proteins and controls (a-TUBB) upon treatment with the indicated epigenetic drugs with for 24 h. (B) Western blot quantification over TUBB control and normalized to DMSO. Each dot represents an independent western blot experiment (n = 2). (C) ABCB1 mRNA levels determined by qRT-PCR and normalized to GAPDH expression levels upon drug addition and RPE-TxR as a control for ABCB1 expression, Error bars show the SD of three technical replicates. (D) pA-DamID Z score signals were quantified using genes as units as Figure 4B (n = 2). Every point represents a single gene; ABCB1 is highlighted in red. Graphs represent the pA-DamID signal correlated between DMSO vs. 5AZA 62.5 nM 72 h- (left) and DMSO vs. GSK126 500 nM 72 h-treated cells (right). Pearson correlation: 0.96 and 0.97, respectively. (E) Crystal violet staining of colony-formation assay under 20 nM of Taxol and the corresponding chromatin drug in RPE-1 iCut WT cells. One million cells were plated per condition. (F) Quantification of the number of Taxol-resistant colonies from (E). Error bars show the average ± SD of independent experiments (n = 3). ns, p > 0.05, Mann- Whitney test.

Article Snippet: Strand-specific libraries were generated using the TruSeq PolyA Stranded mRNA sample preparation kit (Illumina).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Staining, Colony Assay, MANN-WHITNEY

Journal: Cell reports

Article Title: Perturbations in 3D genome organization can promote acquired drug resistance.

doi: 10.1016/j.celrep.2023.113124

Figure Lengend Snippet: Figure 7. Transcription-driven CRISPRa activation of neighboring genes can detach ABCB1 from the NL and lead to Taxol resistance (A) Schematic representation of the Chr7q21.12 region indicating the locations where the sgRNAs were targeting for CRISPRa ABCB1, ABCB4, or RUNDC3B activation. Two regions were independently targeted to upregulate ABCB1: P1 (proximal promoter, 6 sgRNA were used) and P2 (internal promoter, a single sgRNA was used). (B) Local NL detachment caused by CRISPRa ABCB1 or (C) simultaneously CRISPRa ABCB4 and RUNDC3B as analyzed in Figure 4A. Color key domainogram as in Figure 4A. (D) ABCB1 mRNA levels determined by qRT-PCR and normalized to GAPDH upon CRISPRa activation of ABCB1 (P2) or combination of ABCB4 and RUNDC3B. Error bas represent the SD of three independent experiments (n = 3). (E) Crystal violet staining of viability assay on CRISPRa cell lines upon activation of ABCB1 (P2) and the combination of ABCB4 and RUNDC3B. (F) ChIP-qPCR of H3K9me3 and (G) H3K27ac in the ABCB1 regulatory region for CRISPRa WT, ABCB1, or the combination of ABCB4 and RUNDC3B (B4-RUND). TS marks the transcription start site of the promoter. ChIP signal was normalized over input and a positive control specific for each mark. Area fill represents the SEM of six technical replicates coming from two independent experiments.

Article Snippet: Strand-specific libraries were generated using the TruSeq PolyA Stranded mRNA sample preparation kit (Illumina).

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Viability Assay, ChIP-qPCR, Positive Control