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. 2012 Nov 6;3(11):1370–1385. doi: 10.18632/oncotarget.717

Figure 6. Confirmation of ALK4 and MMP11 functions.

Figure 6

(A) Cell lysates prepared from 4T1 cells transiently transfected with miR-98 or GFP and treated with or without Nodal were subjected to Western blot analysis probed with anti-phosphorylated Smad2/3 antibody. Staining for β-actin using the same membranes confirmed equal loading. (B) 4T1 cells transiently transfected with the siRNA or the control oligo were grown on 6-well tissue culture dishes in 5% serum containing medium. Cell proliferation rate was determined by counting the cells on day 1, 3 and 5. * P < 0.01, *P<0.01. Error bars, SD (n = 3). (C) 4T1 cells transiently transfected with the siRNAs or the control oligo were grown on 6-well tissue culture dishes in serum-free conditions. Cell survival was monitored with a light microscope. Surviving cells were harvested and counted. ** P < 0.01. Error bars, SD (n = 3). (D) 4T1 cells stably transfected with miR-98 or GFP were transiently transfected with ALK4 or a control vector followed by culturing in serum-free conditions for 5 days. Cell survival was assayed by counting the viable cells. *P < 0.05. Error bars indicate SEM (n=4). (E) 4T1 cells stably transfected with miR-98 or GFP control vector were transiently transfected with ALK4 or a control vector. The cells were grown on 12-well plates in 5% serum containing medium. The proliferation rate was examined on days 1, 3, and 5. ** P < 0.01. Error bars SEM (n=4). (F) Cell lysates prepared from 4T1 cells transiently transfected with siRNAs targeting MMP11 or a control oligo were analyzed on Western blot probed with anti-MMP11 antibody. (G) 4T1 cells transiently transfected with MMP11 siRNAs or the control oligo (ctrl) were grown on 6-well tissue culture dishes in serum-free conditions. Cell survival was monitored with a light microscope. Surviving cells were harvested and counted. ** P < 0.001. Error bars, SD (n = 10). (H) 4T1 cells transiently transfected with the siRNA or the control oligo. The cells were harvested and suspended in 100 μl serum-free DMEM medium, followed by inoculation onto Matrigel in trans-well inserts. Two days after inoculation, the cells were analyzed for cell invasion. The cells transfected with siRNAs exhibited weaker invasive activity than the cells transfected with a control oligo ** P < 0.001. Error bars, SD (n = 10). (I) Cell lysate prepared from MMP11- or a control vector-transfected 4T1 cells that had been stably transfected with miR-98 or the control GFP were subjected to Western blot analysis probed with anti-MMP11 antibody. Staining for β-actin from the same membrane confirmed equal loading. (J) 4T1 cells stably transfected with miR-98 or GFP were transiently transfected with MMP11 or a control vector followed by culturing in serum-free conditions for 5 days. Cell survival was assayed by counting the viable cells. *P<0.001. Error bars indicate SEM (n=12). (K) 4T1 cells stably transfected with miR-98 or GFP were transiently transfected with MMP11 or a control vector. The cells, harvested and suspended in 100 μl serum-free DMEM medium, were loaded into the Matrigel coated insert and incubated at 37°C for 48 hours fro invasion assay. Expression of MMP11 promoted cell invasion. **, p< 0.001. Error bars indicate SD (n=10).