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. 2020 May 19;42(1):48–57. doi: 10.1093/carcin/bgaa048

Figure 3.

Figure 3.

Effect of Cas/sg RLIP on RLIP gene and protein expression. qPCR using RLIP primers (upper panels), Western blot analysis using antibodies against RLIP (middle panels), and TUNEL assays (lower panels) were performed using MCF7 and MDA-MB231 Cas9/sg RLIP clones (without [control] or with doxycycline treatment, final concentration 0.5 µg/ml) and normalized against β-actin. In qPCR values are means with 95% confidence intervals (n = 4 for each clone); *P < 0.01 and **P < 0.03 compared with respective controls. Data were analyzed using the 2−ΔΔCt method. Effect of Cas9/sg RLIP on apoptosis. MCF7 and MDA-MB231 Cas9/sg RLIP clones without or with doxycycline treatment (0.5 µg/ml, 48 h) were grown on coverslips. TUNEL assays using a Promega fluorescence detection kit were performed using a Zeiss LSM 510 META laser scanning fluorescence microscope with 520 and >620 nm filters. Photographs presented were taken at identical exposure with 400x magnification. Apoptotic cells showed green fluorescence and characteristics of cell shrinkage.