Figure 2.

Effects of interleukin-6 (IL-6) knockdown in 4T1 cells in vitro. (A) Morphology of 4T1^WT, 4T1^NC, and 4T1^IL-6low cells. (B) Cell growth was monitored by Cell Counting Kit-8 assay. (C) Clonogenic survival analysis of infected 4T1 cells. (D) The quantification of the infected 4T1 cell apoptosis was detected using the Annexin V-FITC apoptosis detection kit. Apoptotic cells were recognized by Annexin V⁺PI⁻. (E) Wound healing assay was used to study the migratory ability of infected 4T1 cells (original magnification 40×). (F) The infiltrative and metastatic capability of infected 4T1 cells was evaluated by invasion assay (original magnification 10×). The stained cells from five selected views were observed under a light microscope at 200× magnification. (G) The downstream signaling pathway of IL-6 in 4T1^WT, 4T1^NC, and 4T1^IL-6low cells was detected. The levels of phosphorylated JAK1 (p-JAK1), p-JAK2, p-TYK2, and p-STAT3 were compared using the density ratio of phosphorylated protein to total protein. The levels of the CD126 and gp130 protein were compared using the density ratio of the indicated protein to β-actin. (H) The mRNA levels of gp-130 and CD126 in 4T1^WT, 4T1^NC AND 4T1^IL-6low were determined by RT-PCR.