To monitor equal protein loading RNA polymerase II (RNAPII) and -TUBULIN (-TUB) was detected. cells stably transduced with Dox-inducible retroviral control or Snail1-HA expression vectors. Shown is the mean and SEM; n = 3. (B) Western Blot to analyze Snail1-HA, FOXA1, FOXA1/2, and FOXA3 protein levels upon Dox-induced Snail1-HA expression in HT29 cells. MW = molecular weight in kDa. To monitor equal protein loading RNA polymerase II (RNAPII) was detected. (C) ChIP analysis to test for Snail1-HA occupancy at the promoter in HT29 cells. Data were calculated as percent input. Shown are the mean and SEM; n = 4.(TIF) pgen.1007109.s003.tif (930K) GUID:?2A033242-9A9A-40EE-AF83-F305FE126904 S4 Fig: Snail1 represses promoter activity in LS174T and HT29 cells. (A, B) Luciferase reporter assay in LS174T (A) and HT29 (B) cells with constructs harboring the promoter. Mutations of the respective E-boxes are indicated by red crosses. E-box I apparently has a dual function. It is involved in activation of the FOXA1 promoter in the absence of Snail1-HA. Additionally, E-box I in part mediates the repressive RAF mutant-IN-1 effect of Snail1-HA. Shown is the mean and SEM; n3. RLA: relative luciferase activity. Statistical significance was calculated between samples without and with Snail1 expression.(TIF) pgen.1007109.s004.tif (664K) GUID:?725D4719-07DA-438D-810B-CE676128072F S5 Fig: Expression analyses of genes, and in LS174T cells with wild-type, low, and absent FOXA expression. Expression of and in parental LS174T cells and cell clones subjected to CRISPR/Cas9-mediated genome editing of the FOXA1 locus was assessed by qRT-PCR analyses. rel. expr.: relative expression. Data are shown as mean and SEM; n = 3.(TIF) pgen.1007109.s005.tif (664K) GUID:?982A0EE7-6D55-4571-B7CF-7E44E44CF6EF S6 Fig: Expression and cellular RAF mutant-IN-1 localization of LEF1, CADHERIN11, E-CADHERIN and CLAUDIN3 in LS174T cells with wild-type, low, and absent FOXA expression. Expression of LEF1, CADHERIN11, E-CADHERIN and CLAUDIN3 in parental LS174T cells and cell clones subjected to CRISPR/Cas9-mediated genome editing of the FOXA1 locus was assessed by immunofluorescence studies. Areas within yellow frames were enlarged fivefold and are presented on the right. Scale bars: 50 m and 10 m (fivefold enlargements).(TIF) pgen.1007109.s006.tif (5.7M) GUID:?00A8FA92-134D-4CB6-9080-B96113CC1959 S7 Fig: Significantly higher numbers of FOXA1/FOXA2 binding RAF mutant-IN-1 sites are associated with epithelial gene loci. (A) Total numbers and genic distribution of FOXA1/FOXA2 ChIP-seq peaks in different cell lines. The p-values RAF mutant-IN-1 refer to the results of bootstrapping analyses (exemplary results for this analyses are shown in panel B) to test whether the number of FOXA1/FOXA2 ChIP-seq peaks at epithelial and mesenchymal genes is usually significantly different from random groups of genes. (B) FOXA1 ChIP-seq data from HepG2 cells were analyzed by a bootstrapping approach to estimate whether the number of binding regions at epithelial genes is usually significantly high or low. Out of all 22,000 annotated genes random groups of N = 45 or N = 54 genes representing the sample size of epithelial and mesenchymal gene groups, respectively, were selected, and the numbers of associated peaks were counted. The resulting distribution of associated peak numbers from 10,000 trials is usually shown. The red lines indicate the number of associated peaks for the epithelial and mesenchymal gene groups. The p-values shown were calculated from fitting a skewed normal distribution to the histogram.(TIF) pgen.1007109.s007.tif (1.8M) GUID:?45464626-B1DC-4B52-AB93-1C5E8A6CE3AD S8 Fig: FOXA1/FOXA2 ChIP-seq peaks colocalize more frequently with intergenic enhancer regions at epithelial genes. (A) Genome browser view of the 15-state chromatin model in relation to gene structure and FOXA1/FOXA2 binding regions for mesenchymal (+7.8 kb (A), the +10.0 kb (B) and the ?2.3 kb (C) ECRs. The consensus of the FOXA motifs is usually highlighted by a grey box. Sequence identity is usually indicated by asterisks. The indicated base positions are relative to the transcriptional start site of the RAF mutant-IN-1 human DNA sequence based on the Ensembl genome browser.(TIF) pgen.1007109.s009.tif (2.8M) GUID:?0FF9D137-E968-4E3A-AD7B-9D0ADC072887 S10 Fig: Expression levels of in four CRC cell lines. (A-C) qRT-PCR to analyze expression of (A), (B), and (C) in the indicated CRC cell lines. Data are shown as mean and SEM; n = 3.(TIF) pgen.1007109.s010.tif (211K) GUID:?51E7CB82-4FCA-4485-AED4-7382C9F8208E S11 Fig: Reduced FOXA1 binding to their enhancer regions is paralleled by downregulation of and in Snail1-HA-expressing cells. (A) ChIP analyses to test for binding of FOXA1 to the enhancers in LS174T cells stably transduced with CRF (ovine) Trifluoroacetate Dox-inducible retroviral control or Snail1-HA expression vectors. Data are given as percent input; n = 3. As control (ctrl) the locus at -10.0 kb was analyzed. (B) qRT-PCR to analyze expression of in LS174T cells stably transduced with Dox-inducible retroviral control or Snail1-HA expression vectors. Data are shown as mean and SEM; n = 2. (C) Western Blot.
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