Figure 6

p38-mediated phosphorylation of FOXC2 directly regulates ZEB1 expression. (a) The transcript levels of FOXC2 and ZEB1 in HMLER-vector and HMLER-FOXC2 cells were determined by qRT–PCR, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene to normalize the variability in template loading. Data are reported as mean±s.e.m. (b) Cell lysates from the indicated cells were analyzed by immunoblotting for FOXC2 and ZEB1. β-Actin was used as a loading control. (c) HMLE-Snail and HMLE-Twist cells were immunostained with antibodies against FOXC2 (green) and ZEB1 (red). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (d) HMLE-Snail cells, transduced with control shRNA (shControl) or FOXC2 shRNA (shFOXC2), were immunostained with antibodies against FOXC2 (green) and ZEB1 (red). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (e) The relative expression of ZEB1 mRNA in the indicated cells, transduced with control shRNA (shControl) or FOXC2 shRNA (shFOXC2), was determined by qRT–PCR with GAPDH as the reference gene. Data are reported as mean±s.e.m. (f) The protein levels of FOXC2, ZEB1 and β-actin in the indicated cells, transduced with control shRNA (shControl) or FOXC2 shRNA (shFOXC2), were analyzed by immunoblotting. (g) The relative levels of miR200b and miR200c in HMLER-vector and HMLER-FOXC2 cells were determined by qRT–PCR, with U6 small nuclear RNA as an internal control. Data are reported as mean±s.e.m. (h) The mRNA abundance of miR200b and miR200c in HMLE-Snail cells, transduced with control shRNA (shControl) or FOXC2 shRNA (shFOXC2), was determined by qRT–PCR, with U6 small nuclear RNA as an internal control. Data are reported as mean±s.e.m. (i) The relative levels of ZEB1 transcripts in the indicated cells, treated with vehicle or SB203580, were determined by qRT–PCR, with GAPDH as the reference gene. Data are reported as mean±s.e.m. (j) Cell lysates from the indicated cells, treated with vehicle or SB203580, were analyzed by immunoblotting for ZEB1. β-Actin was used as a loading control. (k) HMLER cells were transduced with empty vector, FOXC2, FOXC2(S367E) or FOXC2(S367A). These cells were treated with vehicle or SB203580, and the corresponding lysates were analyzed by immunoblotting for ZEB1. β-Actin was used as a loading control. (l) A chromatin immunoprecipitation assay was performed using HMLER-FOXC2, HMLER-FOXC2(S367E) and HMLER-FOXC2(S367A) cells to show that FOXC2 binds upstream to the transcriptional start site of the ZEB1 promoter. The y-axis represents the percentage of bound FOXC2, and the x-axis denotes the distance from the ZEB1 transcription start site in kb. n.d., not determined. P-values were calculated using Student's unpaired two-tailed t-test. *P<0.05; **P<0.01; ***P<0.001 compared with the control.