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. 2015 Nov 20;10(11):e0143591. doi: 10.1371/journal.pone.0143591

Fig 5. The effect of HH pathway inhibition on proliferation, viability, migration and SOX18 expression in HeLa cells.

Fig 5

a) The inhibitory effect of cyclopamine on GLI1 and PTCH expression. b) Proliferation curve of HeLa cells. Cells were treated with 10μM cyclopamine or tomatidine as a negative control, and counted after 1, 3 and 5 days of continuous treatment. Results were presented as the means ± SEM of at least three independent experiments. c) MTT viability assay performed after 1, 3 and 4 days of treatment with 10 μM cyclopamine or tomatidine. Relative cell viability was calculated as a percentage of HeLa cells viability after tomatidine treatment that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.d) The effect of cyclopamine on cell’s migration, wound-scratch migration assay. Cells migration was quantified 24 h after scratching in constant presence of cyclopamine or tomatidine,by measuring the difference in gap closure where gap wide at 0 h was set as 100%. Results were presented as the means ± SEM of at least three independent experiments. e) Relative SOX18 expression after cyclopamine treatment detected by qRT-PCR. Relative SOX18 expression was presented as percentage of SOX18 expression in cells treated with tomatidine that was set as 100%. Results were presented as the means ± SEM of at least three independent experiments performed in triplicates. P values were calculated using Student’s t-test, *p ≤ 0.05, **p ≤ 0.01.f) The effect of cyclopamine on SOX18 protein level. Proteins were isolated after three independent treatments together with adequate controls, followed by Western blot. One representative blot was presented. α-tubuline was used as a loading control. The relative SOX18 protein level in HeLa cells upon treatment with cyclopamine was calculated as a percentage of SOX18 level in cells trated with tomatidine which was set as 100%. Data of three independent experiments are presented at histograms as the means ± SEM. Values of p≤0.01 are marked by **.g) The effect of GANT61 on SOX18 protein level. Proteins were isolated after three independent treatments together with adequate controls, followed by Western blot. One representative blot was presented. α-tubuline was used as a loading control.