Figure 1

siRNA-mediated AGR2 knockdown affects anchorage-dependent and anchorage-independent growth in breast cancer cell lines. T47 D, ZR-75-1, MDA-MB-231, and SK-BR-3 cells were transfected with negative control siRNA (iNC), AGR2 siRNA (iAGR2), or untransfected (UT). KSP (DKSP) and its corresponding control (DNC) were used as transfection controls. Results are expressed as a ratio of untransfected cells (±SD), n = 3. (a) Detection of endogenous AGR2 in breast cancer cell line supernatants by IP-Western and whole-cell lysates by Western. AGR2 knockdown was confirmed in lysates 72 hours after transfection. β-Actin served as a loading control. (b) The impact of iAGR2 on anchorage-dependent growth was evaluated at 96 hours after transfection by using the Cell Titer Glo assay. Anchorage-independent growth assays were also used: (i) soft agar colony formation assay (c), with Alamar blue as a readout; (ii) spheroid assay (d), in which lysed spheroid LDH levels were representative of total cell number after 8 days; corresponding spheroid images were also captured. *P < 0.05; **P < 0.01; ***P < 0.001.