Journal: Molecules

Article Title: Effects of Red Sorghum-Derived Deoxyanthocyanidins and Their O-β-D-Glucosides on E-Cadherin Promoter Activity in PC-3 Prostate Cancer Cells

doi: 10.3390/molecules29081891

Figure Lengend Snippet: Effects of the indicated molecules on the activation of the CDH1 promoter. PC-3 prostate cancer cells were transfected with a luciferase plasmid reporter harboring the human −670 to +92 bp CDH1 promoter upstream of the gene encoding Firefly luciferase. Cells were treated with increasing concentrations of the indicated molecules for 6 h. Results for the activation of the CDH1 promoter following treatments of PC-3 cells with P1 ( a ), P2 ( b ), P3 ( c ), P5 ( d ), P7 ( e ), and APN ( f ) are presented. Results are presented as fold activation over the control (DMSO only) (±SEM). A one-way ANOVA was used to analyze data according to the concentrations of indicated molecules, followed by Dunnett’s multiple comparison test for significant differences compared to the control (* p < 0.05).

Article Snippet: The human −670 to +92 bp CDH1 promoter/luciferase reporter construct (#42083) was purchased from Addgene (Watertown, MA, USA).

Techniques: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Control, Comparison

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a – c Scratch assay in MDA-MB-231 and SUM159 cells treated with DMSO or KH-3. a , b Representative images of cell migration at 0 and 24 h after scratching with indicated treatment in MDA-MB-231 ( a ) and SUM159 ( b ) cells, scale bars: 50 μm. c Wound widths in two cell lines 24 h after scratching and treatment (*** P < 0.001, t -test, n = 3). d – f Invasion assay in MDA-MB-231 and SUM159 cells treated by DMSO, KH-3B or KH-3. d , e Representative images of stained invaded cells with indicated treatment in MDA-MB-231 ( d ) and SUM159 ( e ) cells, scale bars: 200 μm. f Invaded cell numbers per image in both cell lines with indicated treatment (*** P < 0.001, one-way ANOVA, n = 6). g The heatmap view of PCR pathway array focusing on invasion and metastasis related genes. The relative mRNA levels were presented as z score, each treatment was triplicated. h CDH1 luciferase reporter assay in HEK 293FT cells treated by DMSO, KH-3B or KH-3. Values are mean ± SD from n = 4 independent experiments (** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: For CDH1 reporter assay, HEK 293FT cells co-transfected with pGL3 vector with or without CDH1 promoter (Addgene, #61798) and renilla were treated with DMSO, KH-3 or KH-3B for 48 h. For FOXQ1 3′-UTR study, MDA-MB-231 and SUM159 cells transfected with pEZX-MT06 reporter vector with or without human FOXQ1 3′-UTR were treated with DMSO, KH-3 or KH-3B at indicated doses for 24 h. The cells were then harvested and assayed using the Dual-Glo Luciferase Assay (Promega).

Techniques: Wound Healing Assay, Migration, Invasion Assay, Staining, Luciferase, Reporter Assay

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a Venn diagram depicting the number of targets identified in two independent RNA-seq experiments. FOXQ1 is a direct HuR target, which is also one of the top mRNAs decreased by KH-3 treatment. b Protein expression levels of HuR, FOXQ1 and E-cadherin in HMEC and a panel of TNBC cell lines. c , d Pull-down analysis of KH-3 disrupting ARE FOXQ1 oligo binding to endogenous HuR in MDA-MB-231 and SUM159 cells. c Representative western blot result from one experiment. d Quantified relative HuR expression. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, *** P < 0.001, one-way ANOVA). e , f RNP IP analysis of HuR bound FOXQ1 mRNA affected by KH-3 in MDA-MB-231 ( e ) and SUM159 ( f ) cells. Values are mean ± SD from three independent experiments (** P < 0.01, *** P < 0.001, one-way ANOVA). g , h Relative FOXQ1 mRNA levels in MDA-MB-231 ( g ) and SUM159 ( h ) cells treated with DMSO, KH-3 or KH-3B at the indicated time points. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA). i , j FOXQ1 3′-UTR luciferase reporter assay in MDA-MB-231 ( i ) and SUM159 ( j ) cells treated by DMSO, KH-3B or KH-3. Values are mean ± SD from n = 3 independent experiments (*** P < 0.001, two-way ANOVA).

Article Snippet: For CDH1 reporter assay, HEK 293FT cells co-transfected with pGL3 vector with or without CDH1 promoter (Addgene, #61798) and renilla were treated with DMSO, KH-3 or KH-3B for 48 h. For FOXQ1 3′-UTR study, MDA-MB-231 and SUM159 cells transfected with pEZX-MT06 reporter vector with or without human FOXQ1 3′-UTR were treated with DMSO, KH-3 or KH-3B at indicated doses for 24 h. The cells were then harvested and assayed using the Dual-Glo Luciferase Assay (Promega).

Techniques: RNA Sequencing, Expressing, Binding Assay, Western Blot, Luciferase, Reporter Assay

Journal: Communications Biology

Article Title: Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

doi: 10.1038/s42003-020-0933-1

Figure Lengend Snippet: a , b Invasion assay in parental MDA-MB-231 cells, sgControl and two HuR KO clones transfected with control vector or vector containing FOXQ1 cDNA. a Representative images of stained invaded cells, scale bars: 200 μm. b The number of invaded cells per image (*** P < 0.001, one-way ANOVA, n = 6). c mRNA expression levels of FOXQ1, CDH1 and CD82 in parental MDA-MB-231 cells, sgControl and two HuR KO clones transfected with control vector or vector containing FOXQ1 cDNA. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, *** P < 0.001, two-way ANOVA). d , e Invasion assay in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with DMSO or 10 μM KH-3 treatment. d Representative images of stained invaded cells, scale bars: 200 μm. e The number of invaded cells per image (*** P < 0.001, one-way ANOVA, n = 6). f mRNA expression levels of FOXQ1, CDH1 and CD82 in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with treatment of DMSO or 10 μM KH-3. Values are mean ± SD from n = 3 independent experiments (** P < 0.01, *** P < 0.001, two-way ANOVA). g , h Protein expression levels of FOXQ1, Bcl-2, Msi2, β-catenin, and HuR in MDA-MB-231 cells transfected with control vector or vector containing FOXQ1 cDNA together with treatment of DMSO or KH-3 at the indicated doses for 48 h. α-Tubulin is used as loading control. g Representative western blot results from one experiment. h Quantified relative expression of HuR and downstream targets. Values are mean ± SD from n = 3 independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA).

Article Snippet: For CDH1 reporter assay, HEK 293FT cells co-transfected with pGL3 vector with or without CDH1 promoter (Addgene, #61798) and renilla were treated with DMSO, KH-3 or KH-3B for 48 h. For FOXQ1 3′-UTR study, MDA-MB-231 and SUM159 cells transfected with pEZX-MT06 reporter vector with or without human FOXQ1 3′-UTR were treated with DMSO, KH-3 or KH-3B at indicated doses for 24 h. The cells were then harvested and assayed using the Dual-Glo Luciferase Assay (Promega).

Techniques: Invasion Assay, Clone Assay, Transfection, Control, Plasmid Preparation, Staining, Expressing, Western Blot

Journal: Nature Communications

Article Title: Switch-like enhancement of epithelial-mesenchymal transition by YAP through feedback regulation of WT1 and Rho-family GTPases

doi: 10.1038/s41467-019-10729-5

Figure Lengend Snippet: Switch-like regulation of YAP in expanding epithelial layers loosening cell–cell adhesion. a Cells on flat substrata and NRA analyzed by immunoblotting using YAP and phosphorylated YAP antibodies. b Cell migration speed of individual YAP KD and YAP OE cells in cell sheets as a function of the distance from the sheet edge on NRA (each number of independently analyzed cells, n , is indicated, * = statistical significance of speed of control vs. YAP KD cells on NRA (green) and of control vs. YAP OE cells on NRA (purple), * P < 0.05, ** P < 5 × 10 −4 , and *** P < 5 × 10 −6 ). c Immunofluorescence staining for YAP and active β-catenin in epithelial cell sheets on flat substrata and NRA. Translocation of YAP and active β-catenin into nuclei observed in marginal zones and FLPs of sheets expanding on NRA (brown boxes) and mostly cytoplasmic YAP and active β-catenin localization in submarginal cells on NRA (red boxes) and on flat substrata. The samples were analyzed after 8 h from the initiation of sheet expansion. d Fractions of nuclei displaying different intensities of YAP and active β-catenin staining as a function of the distance from the sheet edge determined at 8 h after initiation of sheet expansion (the edge is coincident with the concave regions at the bases of FLPs in panel c ). e Velocity correlation length in y-direction, parallel to the direction of epithelial expansion for the control and YAP KD cells on flat substrata and NRA (each number of independently analyzed cells, n , is indicated, * = statistical significance of correlation length, * P < 0.05, ** P < 0.01, and n.s = no significance). f Cell migration speed of individual cells in cell sheets as a function of the distance from the sheet edge on flat substrata and NRA, in the presence of an E-cadherin functional blocking antibody. Dashed line with a square marker indicates the average cell migration speed of isolated control cells and solid line with a circle marker corresponds to that of isolated YAP KD cells (each number of independently analyzed cells, n, is indicated, # = statistical significance of speed values in the marginal region vs. the most submarginal region of YAP KD cells with drugs, ### P < 5 × 10 −6 . * = statistical significance of control vs. YAP KD cells with drugs (green) and of YAP KD cells with vs. without drugs (black), * P < 0.05, ** P < 5 × 10 −4 , and *** P < 5 × 10 −6 ). g Control, YAP KD , and YAP OE cells were immunoblotted using active β-catenin and E-cadherin antibodies. All error bars are S.E.M and statistical significance was determined by two-sided Student’s t -test

Article Snippet: The CDH1 (E-cadherin) promoter luciferase activity reporter vectors were purchased from Addgene (#42081).

Techniques: Western Blot, Migration, Control, Immunofluorescence, Staining, Translocation Assay, Functional Assay, Blocking Assay, Marker, Isolation

Journal: Nature Communications

Article Title: Switch-like enhancement of epithelial-mesenchymal transition by YAP through feedback regulation of WT1 and Rho-family GTPases

doi: 10.1038/s41467-019-10729-5

Figure Lengend Snippet: YAP regulates E-cadherin through WT1 in epithelial layers on NRA. a Immunofluorescence staining for WT1 in YAP KD cell sheets (top), and for YAP in WT1 KD cell sheets cultured on NRA. The samples were fixed after 8 h to remove stencils. b Co-IP analysis using the YAP antibody, followed by immunoblotting using the WT1 antibody. c Chromatin immunoprecipitation (ChIP) analysis of the WT1-YAP complex binding at the E-cadherin promoter ( n = 3). We extracted cross-linked chromatin and immunoprecipitated it using antibodies against YAP, WT1, IgG (negative control), and RNAPII or histone H3 (see Supplementary Fig. ) antibodies (positive controls). The immunoprecipitated chromatin and input genomic DNA were used for amplification of the E-cadherin promoter, CTGF promoter (known to be regulated by YAP but not WT1, and used as another control), and GAPDH promoter ( n = 4 biologically independent samples, * = statistical significance of PCR products from each sample vs. IgG, * P < 0.05 and ** P < 0.01). d E-cadherin expressions of control and WT1 KD cells analyzed by immunoblotting with WT1 and E-cadherin antibodies ( n = 3 biologically independent samples, * = statistical significance of E-cadherin expression of control and WT1 KD cells, *** P < 5 × 10 −3 ). e Cell migration speed of individual cells in control and WT1 KD epithelial cells in the marginal region of the sheets in the presence of an E-cadherin blocking antibody (each number of independently analyzed cells, n , is indicated, * = statistical significance, n.s = no significance, and *** P < 5 × 10 −3 ). f Dissemination of cells in control and WT1 KD epithelial sheets on NRA in the presence of an E-cadherin blocking antibody ( n = 4 biologically independent experiments, * = statistical significance, * P < 0.05, *** P < 5 × 10 −3 , and n.s = no significance). g Immunoblotting of phosphorylated YAP and total YAP in cell sheets cultured in the presence of different concentrations of E-cadherin blocking antibody ( n = 3 biologically independent samples). h Schematic of the YAP-mediated cell dissemination triggered by mechanical cues stemming from NRA, operating through E-cadherin control by YAP-WT1 complexes and leading to cell dissemination in epithelial cell sheets on NRA. All error bars are S.E.M and statistical significance was determined by two-sided Student’s t -test

Article Snippet: The CDH1 (E-cadherin) promoter luciferase activity reporter vectors were purchased from Addgene (#42081).

Techniques: Immunofluorescence, Staining, Cell Culture, Co-Immunoprecipitation Assay, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Amplification, Control, Expressing, Migration, Blocking Assay

Journal: Nature Communications

Article Title: Switch-like enhancement of epithelial-mesenchymal transition by YAP through feedback regulation of WT1 and Rho-family GTPases

doi: 10.1038/s41467-019-10729-5

Figure Lengend Snippet: Double-feedback loop with YAP activated on NRA regulating EMT. a Simulation result showing that feedback mechanisms lead to emergence of two stable states of high and low YAP activity, indicating epithelial (low Rac1, high E-cadherin, and low YAP) and partial EMT (high Rac1, low E-cadherin, and high YAP) states depending on Rac1 and E-cadherin activity in this system. b Cell migration speed on NRAs with different rigidity values, suggesting that rigidity can regulate the basal rate of YAP activation at different initial distances from the edge of the sheet (all error bars are S.E.M, each number of independently analyzed cells, n , is indicated, * = statistical significance of the speed of cells on NRA in which the rigidities are 10 MPa vs. 1 GPa, * P < 5 × 10 −2 , ** P < 1 × 10 −2 , and *** P < 5 × 10 −3 , all two-sided Student’s t -test). c Simulated bimodal distribution of YAP activity as a function of the distance from the sheet edge on NRA substrata of different rigidity values. d Rigidity-dependent YAP localization in nuclei of cells cultured on NRA. Immunofluorescence staining of YAP showing nuclear localization at different distances from the sheet edge on NRAs with different rigidity values (top). Fractions of nuclei displaying different intensities of YAP staining as a function of the distance from the sheet edge on NRA having different rigidity (bottom) (see details in Supplementary discussion and Supplementary Fig. ). The samples were fixed after 6 h to remove stencils and induce epithelial expansion. e Schematic description of the regulation of EMT by YAP-mediated topographically induced mechanical input, showing full EMT in marginal cells exposed to cell-free areas at the fronts of FLPs, partial EMT in extensive marginal areas, and epithelial organization (no EMT) in the areas most distant from the edge

Article Snippet: The CDH1 (E-cadherin) promoter luciferase activity reporter vectors were purchased from Addgene (#42081).

Techniques: Activity Assay, Migration, Activation Assay, Cell Culture, Immunofluorescence, Staining