Figure 5. Six1 knockdown decreases distant metastasis, and the Six family member, Six2, likely compensates for loss of Six1.

(A) Diagrammatic representation of the metastasis experiment performed in A–C, in which tumors were allowed to grow for the same amount of time before sacrifice. 66cl4-scramble or 66cl4-Six1 KD1/2 cells were injected into the fourth mammary fat pad of Balb/c mice, and metastases were measured weekly. (B) Representative bioluminescent imaging of Balb/c mice 40 days after injection of 66cl4-scramble or 66cl4-Six1 KD1/2 cells into the fourth mammary fat pad. Quantitation of distant luminescent signal, likely in lungs (yellow boxed region), in 66cl4-scramble and Six1 KD groups. p/s, photons per second. (C) Clonogenic assays were used to detect metastases in lungs of animals bearing 66cl4-scramble and 66cl4-Six1 KD tumors. Representative images from 66cl4-scramble and 66cl4-Six1 KD are shown. (D) Diagrammatic representation of the metastasis experiment performed in D–G, in which tumors were allowed to grow to the same size before sacrifice. 66cl4-scramble or 66cl4-Six1 KD1/2 cells were injected into the fourth mammary fat pad of Balb/c mice, and metastases were measured when tumor sizes were comparable. (E) Quantitation of bioluminescent imaging (photons per second) emanating from the region of lungs in Balb/c mice injected with 66cl4-scramble or 66cl4-Six1 KD1 and KD2 when tumors reached comparable sizes (day 46 for 66cl4-scramble and day 54 for 66cl4-Six1 KD1 and KD2). (F) Six1 expression was measured by real-time PCR in parental Six1 KD2 cells (in culture), Six1 KD2 cells retrieved from lungs of 3 individual mice, and 66cl4-scramble control cells retrieved from lung of 1 animal. The numbers over the bars represent Six2 expression fold change over the value of Scram. (G) Six2 expression was measured by real-time PCR in the above-mentioned cells. *P < 0.05; **P < 0.01.