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: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hepatocytes were transfected with Igfbp1-promoter/pGL3 (Addgene, cat # 12146) and Gfp-FoxO1 plasmids (Addgene, cat # 17551), and renilla luciferase vector was co-transfected as an internal control (Addgene, cat # 27163) ( ; ; ).

Techniques: Virus, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Reporter Assay, Negative Control, Luciferase, Software

Journal: Nature Communications

Article Title: Optimal myelin elongation relies on YAP activation by axonal growth and inhibition by Crb3/Hippo pathway

doi: 10.1038/ncomms12186

Figure Lengend Snippet: ( a ) Western blots of differentiated primary rat Schwann cells following infection with GFP control or constitutively active YAP S127A Flag adenovirus. GFP and Flag panels confirm infection in four independent cultures of each condition. Myelin proteins EGR2 (∼50 kDa) expression is increased, MPZ (∼27 kDa) is downregulated and MBP (∼45 kDa) is unchanged by YAP activation. A positive control for MBP from whole sciatic nerve extract is shown in insert, right. MAG (∼100 kDa) is also upregulated as is expression of proteins with relevance to myelination, Rab11a (∼20 kDa) and Laminin1 (∼175 kDa), in response to YAP activation. Actin (∼48 kDa) was used to verify loading. ( b ) Quantification and statistical analysis of the change in selected protein expression in primary RSCs following differentiation and infection with dominant active YAP S127A, relative to levels in cells infected with GFP control (normalized to 1, dotted line). ( c ) HEK 293T cells were transfected with plasmids expressing Mpz or Egr2 promoter driving Firefly luciferase expression, and either GFP, YAP alone or YAP and TEAD isoforms together. Luciferase levels were normalized to cells transfected with control GFP (three independent experiments in triplicates). Statistical analysis compares each transfection with the GFP infection for each promoter tested. a.u., arbitrary units. Statistical two-tailed Student's t -tests: * P <0.05; ** P <0.01; *** P <0.001; NS, P >0.05 not significant (NS). Error bars show s.e.m.

Article Snippet: Forty-eight hours later, cells were transfected in Optimem medium (Invitrogen) with 0.5 μl Lipofectamine 2000 (Invitrogen) and 0.6 μg DNA: 150 ng Renilla luciferase construct (pRL-CMV-Renilla, Promega), 150 ng Firefly luciferase constructs (pGL3 MPZ intron1 Addgene 21630 or pGL3Krox20 promoter Addgene 21260), 150 ng overexpressing construct 1 (pEGFP Clontech used as control, p2xFLAGhYAP1, Addgene 17791) and 150 ng of overexpressing construct 2 (pBluescript plasmid as carrier; pRK5-Myc-TEAD1 Addgene 33109; Myc-TEAD4 Addgene 24638).

Techniques: Western Blot, Infection, Control, Expressing, Activation Assay, Positive Control, Transfection, Luciferase, Two Tailed Test