LGR5 Antibody - #DF2816

Figure 5. The regulatory role of PHGDH in maintaining the stemness of CRC cells. (A) Western blot analysis of PHGDH protein level in HCT-116 cells with stable expression of scrambled or PHGDH shRNA, and in parallel or PHGDH overexpression HCT-15 cells. (B) Representative micrographs of tumorspheres from HCT-116 cells bearing scrambled or PHGDH shRNA (up) or PHGDH-overexpression HCT-15 cells (down). (C) Quantification of the numbers of tumorspheres in (B). (D) Limiting dilution analysis (LDA) to assess the sphere-initiation capacity of stable PHGDH knockdown or overexpression cells. Cells were planted into 96-well plates at densities of 8, 15, 31, or 62 cells per well and cultured for 10 days, followed by counting of wells containing tumorspheres and analysis utilizing ELDA software. (E) Representative images of colony formation in soft agar. (F) Quantification of colony numbers in (E). (G) Quantitative RT-PCR analysis of CRC stem cell markers (LGR5, CD133, CD44, and CD166). (H) Detection of PHGDH protein levels of CD44 and LGR5 by Western blot. (I) Limiting dilution tumorigenesis assays conducted by subcutaneously injecting scrambled or PHGDH shRNA cells (1 × 104, 1 × 105, and 1 × 106) into BALB/c nude mice. After 39 days, animals were sacrificed. The ratio of formed tumors to the total number in each group is presented in a table and illustrated in a pie chart. (J) Images of tumors excised from mice injected with scrambled or PHGDH shRNA cells. (K) Tumor tissue weight. (L) Tumor volume growth curve for each group. (M) Western blot analysis of CD133, CD44, LGR5 and CD166 level in sh-PHGDH group and NC group. Statistical significance was determined by unpaired t-test. Error bars denoted SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 4. Expression of the epithelial cell marker EpCAM and the stem cell marker LGR5 in the spheres. Both EpCAM (A-C) and LGR5 (D-F) were extensively expressed in the spheres. Ab (-), without application of the first antibody as a negative control (G-I). Scale bars = 25 μm.