Fig 1: Transfection of synthetic guide RNAs is effective to generate potent gene knockouts for cell biological analyses. (A, B) Fluorescence images of cells with RNA guides targeting KIF11 (A) or CENPN (B), stained for DNA and/or microtubules as indicated. Mitotic index indicates the percentage of total cells in mitosis. For KIF11, Cas9 was induced for 66–68 h before imaging and the synthetic/transcribed RNAs were transfected 48 h before imaging and analysis. For CENPN, both Cas9 induction and RNA transfection were performed 60 h before imaging and analysis. (C–E) Quantification of knockout phenotypes for KIF11 (C) and CENPN (D) by immunofluorescence and RELA (E) by flow cytometry. The number of cells scored and statistical comparisons performed can be found in Supplemental Tables S1 and S2, respectively. Data are displayed as mean with error bars indicating SD. (F) Immunofluorescence images of cells with RNA guide targeting RELA and stained for RELA (cyan) and microtubules (red). Cas9 expression was induced for 84 h before imaging and the synthetic/transcribed RNAs were transfected 72 h before imaging and analysis. Scale bar = 20 µm (A, B) or 100 µm (F).
Fig 2: Reverse transfection allows real-time imaging of depletion phenotypes. Time-lapse stills of HeLa cells seeded on a printed sgRNA array with control spots (left) or spots containing an sgRNA targeting KIF11 (right). Spots were identified using a fluorescence dye-conjugated RNA (channel not shown). Cas9 expression in these cells was induced 12–18 h before seeding. Cells were stained for DNA and imaged in a heated microscope chamber at the indicated time postseeding. Dotted circles (orange) indicate the region of the printed spot. Red arrowheads highlight cells with monopolar spindles. Scale bar = 100 µm.
Fig 3: Comparison of efficiency of crRNAs and sgRNAs for gene disruption. (A, B) Quantification of knockout phenotype for cells transfected with 15 nM synthetic crRNA or sgRNA targeting KIF11 (A) or CENPN (B), imaged at the indicated times posttransfection. Mitotic index indicates the percentage of total cells in mitosis. Cas9 expression was induced 18–20 h before transfection for KIF11 or simultaneously with RNA transfection for CENPN. Number of cells counted and statistical comparisons performed can be found in Supplemental Tables S1 and S2. (C–E) Titration of RNA guide concentrations for crRNA and sgRNA targeting KIF11 (C), CENPN (D), and RELA (E). Cells transfected with KIF11-targeting crRNA and sgRNA were treated as described above and imaged at 48 and 30 h posttransfection, respectively, to catch the peak knockout period for each synthetic guide. Cells transfected with CENPN or RELA RNA guides were treated as described in Figure 1 and analyzed at 60 or 72 h posttransfection, respectively. Quantification of the knockout phenotypes was done using immunofluorescence (KIF11/CENPN) and flow cytometry (RELA). Data are displayed as mean with error bars indicating SD.
Fig 4: Reverse transfection allows efficient delivery of sgRNAs for phenotypic analyses. Immunofluorescence images of HeLa cells that were fixed and imaged 72 h postseeding on printed sgRNA array with control spots or spots containing sgRNA targeting KIF11 (A) or RELA (B). Cas9 expression in these cells was induced 12–18 h before seeding. For the KIF11 sgRNA array, cells were stained for DNA (cyan) and microtubules (red). Dotted boxes (white) indicate magnified region (right). For the RELA sgRNA array, cells were stained for RELA (cyan) and microtubules (red). In all images, the dotted circles (orange) indicate region of printed spot. Scale bar = 100 µm.
Fig 5: Fas, p21 and Eg5 mediates CHK1's effect on ER+/PR+/HER2- cell survival. A-B Flow cytometry was performed to assess the effects of CHK1 suppression on cell cycle distribution. A significant S phase decrease and increase of G0/G1 and G2/M phase appeared in MCF-7 cells (A). No significant difference was found in MDA-MB-231 cells (B). C We also analyzed cell apoptosis by flow cytometry. We found that CHK1 inhibition induced apoptosis in MCF-7 cells. D Heatmap of differentially expressed genes (log2 [fold change] > 0.5 or < -0.5, P < 0.05) between the si-CHK1 and si-control groups in MCF-7 cells from GSE31912. E After GO enrichment analysis of the top 100 differentially expressed genes in GSE31912, we selected the GO terms and genes of interest as potential targets. F Outside of for the top 100, 7 targets were shared between differentially expressed genes from GSE31912 and genes co-expressed with CHK1. G Western blot results showed that with CHK1 suppressed, Fas and p21 were upregulated while Eg5 was downregulated in MCF-7 and T47D cells. H The role of CHK1 varies with ER/PR status in targeted therapy for breast cancer. When ADR-activated CHK1 is inhibited in ER-/PR-HER2- breast cancer, the pro-apoptotic effects mediated by MSX2 and BIM are enhanced, due to the loss of cell cycle arrest mediated by the MCC-APC/C-cyclin B1 axis. However, in ER+/PR+/HER2- breast cancer, the suppression of CENPF-mediated transcriptional activation for CHK1 is induced by ADR itself. The role of CHK1 inhibition is reversed, so that it shows the single-agent antitumor activity mediated by p21, Eg5 and Fas. Data shown represent the means (±- SD) of three independent experiments; *P < 0.05, **P < 0.01; NS, not significant; one-way ANOVA (A-C) or Bayes's test (D). si-up: genes upregulated in si-CHK1 group; si-down: genes downregulated in si-CHK1 group; Co-N: negatively associated with CHK1 in the co-expression level; Co-P: positively associated with CHK1 in the co-expression level.
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