Fig. 1
Assessment of CRISPRi/a in activation of endogenous FOXP3 and repression of XIST in human breast cancer cells. A efficacy of co-transduction of the XIST (mIFP) and FOXP3 (mCherry) sgRNAs in CRISPRi/a MDA-MB-231 cells before and after Dox addition at days 0, 2, 4, and 6 as determined by fluorescence microscopy. The expression of SpdCas9-KRAB (GFP) is induced by Dox in CRISPRi/a cells. BF, bright field. Scale bar, 1,000 μm. B protein expression of SpdCas9 and SadCas9 in CRISPRi/a MDA-MB-231 cells before and after sgRNA transduction and Dox treatment at days 0 and 4 as determined by Western blots with specific anti-SadCas9 and anti-SpCas9 antibodies. C, D quantitative expression analysis of FOXP3 and XIST before and after sgRNA transduction and Dox treatment of CRISPRi/a MDA-MB-231 cells at days 0, 2, 4, 6, 8, and 10 as determined by qPCR. The fold change in mRNA expression was calculated using the 2-ΔΔ Ct method with GAPDH mRNA as an internal control. E targeted cell sorting of the CRISPRi/a HCC202 cells after transduction of XIST (mIFP) and FOXP3 (mCherry) sgRNAs and addition of Dox at day 4 as determined by flow cytometry. F, G quantitative expression analysis of FOXP3 and XIST before and after sgRNA transduction and Dox addition in CRISPRi/a HCC202 cells at days 0, 2, 4, 6, 8, and 10 as determined by qPCR. H expression of XIST in CRISPRi/a cells after Dox addition for 4 days as determined by RNA fluorescence in situ hybridization analysis. Cells were hybridized to the XIST probe (green). DAPI (blue) was used as a nuclear counterstain. DAPI, 4’,6-diamidino-2-phenylindole. Scale bar, 50 μm. I mutation analysis of a 798G/C mutation (red arrow) of the human FOXP3 transcript in CRISPRi/a HCC202 cells before and after activation of FOXP3 as determined by cDNA sequencing. J effect of CRISPRi/a-induced endogenous FOXP3 on growth of HCC202 cells. Cell proliferation was measured after Dox treatment. K bioluminescent and optical images of CRISPRi/a (with FOXP3/XIST sgRNAs) and scrambled (CRISPRi/a without FOXP3/XIST sgRNAs) HCC202 cells using the In Vivo Bio-luminescence Imaging System. Eight-week-old NSG female mice were performed by intratibial injection with 1x105 CRISPRi/a and scrambled HCC202 luciferase cells, respectively, followed by Dox injection (2.5 mg/kg weekly). L Quantitative analysis of the ex vivo bioluminescent imaging results from panels in K. M Representative optical images of lung metastasis in mice with CRISPRi/a and scrambled control cells at day 28 after bone transplantation. Red down arrows indicate Dox injection. N Quantification of lung metastatic nodules. The number of surface tumor lesions over all lobes of the lungs was scored for metastatic nodules. Data are presented as the means ± standard deviation. p values were determined by ANOVA followed by Tukey's post hoc test, Mann Whitney test or by two-way ANOVA test vs. control group. All experiments were repeated three times