The experiment that is shown in Figure 3b was doe with limiting-dilution analyse

Question

The experiment that is shown in Figure 3b was doe with limiting-dilution analyses (LDAs) to get the results in Figure 3c. What further conclusions did the researchers make about BMI-1 based on the results? What else could the researchers learn about BMI-1 from the experiments that are shown in figure 3e to get the results in figure 3f?   

BMI-1-KD impairs colorectal CIC self-renewal In addition to the reduction in tumor mass of BMI-1-KD cells, there was also a decrease in the total number of tumors initiated, suggesting that BMI-1 may regulate colorectal CICs (Fig. 3a). To enumerate the frequency of CICs directly, we employed clonal tumor initiation assays performed with limiting-dilution analyses (LDAs) (Fig. 3b). BMI-1 knockdown decreased the frequency of tumor-initiating cells 12-fold in LS174T cells (P < 0.0001, Fig. 3c and Supplementary Table 1). In four primary samples, the frequency was 11-, 65-, 19- and 54-fold lower in the BMI-1-KD groups as compared to controls (Fig. 3c and Supplementary Table 1). These quantitative studies establish that BMI-1 knockdown prevents tumor initiation, thereby functionally lowering the frequency of CICs. To directly determine whether BMI-1 was affecting CIC selfrenewal, we took two additional approaches. We employed the widely used in vitro assay of sphere initiation but used LDA to ensure the accuracy of our measurements. We determined that there was a 283- to 661-fold decrease in the sphere re-initiating cell frequency in the BMI-1-KD group compared to controls (Fig. 3d and Supplementary Table 2). We observed similar results with two additional shRNAs (Supplementary Fig. 8). Therefore, BMI-1 knockdown severely abrogates the self-renewal capacity of colon cancer cells in vitro. The definitive method to assess the self-renewal potential of CICs is to carry out secondary tumor formation assays in vivo using tumors initiated by a single CIC (see details in Supplementary Methods). We therefore injected shLUC and shBMI-1 cells from three samples at limiting doses into mice; we transplanted tumors generated from a single colorectal CIC into secondary recipients using limiting dilution (Fig. 3e). There was a 60-fold decrease in the frequency of selfrenewing CICs in the BMI-1-KD group in LS174T cells (P < 0.0001, Fig. 3f). For sample 01, the frequency of CICs in the BMI-1-KD group was reduced eightfold (P = 0.005), and for sample 02, we observed a 15-fold reduction in CICs (P < 0.0001, Fig. 3f and Supplementary Table 3). Taken together, these findings indicate that BMI-1 silencing leads to a considerable decrease in the number of colorectal CICs in primary human tumors.

Figure 3 BMI-1 knockdown reduces the frequency of self-renewing colorectal CICs. (a) Representative image of tumors generated in mice that were injected with cells described in Figure 1d. (b) Schematic illustrating the experimental approach taken to assay the frequency of CICs after BMI-1 knockdown (see Online Methods). (c) CIC frequency of shBMI-1– or shLUC-transduced cells as measured by LDA in vivo. Data are expressed as mean and 95% confidence interval (CI). (d) The number of secondary sphere-forming units for the shLUC and shBMI-1 groups. Data are expressed as mean and 95% CI. (e) Schematic representation of the experimental approach taken to quantify the self-renewal ability of a single colorectal CIC in vivo. (f) Frequency of secondary CICs in vivo. Data are expressed as mean and 95% CI. Frequency and probability estimates were computed using the ELDA software (**P < 0.01 and ***P < 0.001).

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The experiment that is shown in Figure 3b was doe with limiting-dilution analyse

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