flag snai1  (Addgene inc)

Journal: bioRxiv

Article Title: TANGO-Light - optogenetic control of transcriptional modulators

doi: 10.1101/2023.05.31.543150

Figure Lengend Snippet: ( A ) Diagram showing the architecture of the TL-SNAI1 system. ( B ) Immunocytochemical staining of SNAI1 (green) before (DARK) and after photoactivation pattern of 0.05s activation and 5 s brake (LIGHT), showing the relocalisation of SNAI1 from the membrane to the nucleus. Hoechst 33,342 (blue) was used to stain nuclei, ( C ) MDCK-2 cells stained with Vybrant™ DiD (purple) dye and Hoechst 33,342 (blue) before and after blue illumination: TL-SNAI1, mock transfected, as a negative control, and transfected with SNAI1 as a positive control. (D) The mRNA expression of MMP9 , VIM , CDH1 , FN1 , ZEB1 , and STAT3 genes was determined by qPCR (2–ΔΔCt) 16 after light stimulation of TL-SNAI1 H2170. Some of the light-activated cells were additionally cultured with TGFβ in medium. The results are presented as fold change (FC) values (mean□±□SD) normalized to the GAPDH gene expression. All data were analyzed with a one-way ANOVA test and Tukey’s multiple comparisons post-hoc test vs. not light stimulated cells (the time point 0 h and D; dark, respectively). (E) TL-SNAI1 H2170 organoids were cultured between two layers of Matrigel (lower gel − 4 mg/ml and upper gel − 2 mg/ml) with TGFβ-supplemented in the gel and medium of the top of spheroids and controls without this EMT inducer. Spheroids were imaged for 9 days. Cells were light-activated using short pulses for 3 h every day (LIGHT). Images represents comparison of the morphology of spheroids formed by TL-SNAI1 H2170 photo-stimulated (LIGHT) or kept in the dark (DARK).

Article Snippet: KOD-Xtreme hot-start DNA polymerase (Merck Millipore), DreamTaqTM Green PCR Master Mix (ThermoFisher Scientific), DpnI restriction enzyme (NEB), Gibson Assembly® Master Mix (NEB), ampicillin (BRAND), DNA Clean & Concentrator and Zyppy Plasmid Kits (Zymoresearch), TurbofectTM Transfection Reagent (ThermoFisher Scientific), Lipofectamine 3000TM (ThermoFisher Scientific),VybrantTM DiD Cell-Labeling Solution (ThermoFisher Scientific), Bright-Glo Luciferase Assay System (Promega), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (PromoCell), plasmids pCDNA3.1(+)-CMV-βarrestin2-TEV (Addgene plasmid #107245; http://n2t.net/addgene:107245 ; RRID:Addgene_107245) and Flag Snai1 6SA (Addgene plasmid #16221; http://n2t.net/addgene:16221 ; RRID:Addgene_16221), plasmid pDONR223_NOTCH1_ICN (Addgene plasmid #82087; http://n2t.net/addgene:82087 ; RRID:Addgene_82087), plasmid GPCR-TANGO and HTLA cells were a gift from the lab of Richard Axel, Howard Hughes Medical Institute, Department of Biochemistry and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, plasmid PA-CXCR4 was a gift from the lab of Minsoo Kim, Ph.D, University of Rochester Medical Center School of Medicine and Dentistry.

Techniques: Staining, Activation Assay, Membrane, Transfection, Negative Control, Positive Control, Expressing, Cell Culture, Gene Expression, Comparison

Journal: bioRxiv

Article Title: TANGO-Light - optogenetic control of transcriptional modulators

doi: 10.1101/2023.05.31.543150

Figure Lengend Snippet: (A) Diagram showing the structure and action of a light activated system. (B) Light activation of TL-N1ICD, resulted in a dose-dependent increase of FFluc expression as compared to those kept in the dark. (C) In order to select the optimal length of irradiation, cells were pulse-irradiated for 1, 3, 6, 12, 24 and 48 h. Cells were photoactivated 24 h after transfection and lysed 48 h after the start of irradiation to measure FFluc activity. The reporter’s response was significantly higher for longer exposure times. (D) Immunocytochemical staining of N1ICD (green) before (DARK) and after photoactivation (LIGHT), showing the relocalisation of NOTCH1 from the membrane to the nucleus. Hoechst 33,342 was used to stain nuclei (blue). (E) The mRNA expression of Notch1-target genes: HES1 , SNAI1 , FN1 (FN1), VIMENTIN (VIM) and SLUG was determined by qPCR (2–ΔΔCt) 24 after light stimulation of TL-N1CD HEK293T cells. The results are presented as fold change (FC) values (mean□±□SD) normalized to the GAPDH gene expression. (F) 3D organoid images of MDA-MB-468 cells in 9th day not photostimulated (DARK) and photostimulated (LIGHT). Light stimulation resulted in a noticeable increase in the growth of the spheroids, and morphological changes. (G) Comparison of the growth MDA-MB-468 organoids in dark and light-activating condition for 9 days. All data were analyzed with a one-way ANOVA test and Tukey’s multiple comparisons post-hoc test vs. not light stimulated cells (the time point 0 h and D; dark, respectively). *p□ ≤ □0.05; **p□ ≤ □0.01; ***p□ ≤ □0.001 were considered statistically significant, ns – non significant.

Article Snippet: KOD-Xtreme hot-start DNA polymerase (Merck Millipore), DreamTaqTM Green PCR Master Mix (ThermoFisher Scientific), DpnI restriction enzyme (NEB), Gibson Assembly® Master Mix (NEB), ampicillin (BRAND), DNA Clean & Concentrator and Zyppy Plasmid Kits (Zymoresearch), TurbofectTM Transfection Reagent (ThermoFisher Scientific), Lipofectamine 3000TM (ThermoFisher Scientific),VybrantTM DiD Cell-Labeling Solution (ThermoFisher Scientific), Bright-Glo Luciferase Assay System (Promega), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (PromoCell), plasmids pCDNA3.1(+)-CMV-βarrestin2-TEV (Addgene plasmid #107245; http://n2t.net/addgene:107245 ; RRID:Addgene_107245) and Flag Snai1 6SA (Addgene plasmid #16221; http://n2t.net/addgene:16221 ; RRID:Addgene_16221), plasmid pDONR223_NOTCH1_ICN (Addgene plasmid #82087; http://n2t.net/addgene:82087 ; RRID:Addgene_82087), plasmid GPCR-TANGO and HTLA cells were a gift from the lab of Richard Axel, Howard Hughes Medical Institute, Department of Biochemistry and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, plasmid PA-CXCR4 was a gift from the lab of Minsoo Kim, Ph.D, University of Rochester Medical Center School of Medicine and Dentistry.

Techniques: Activation Assay, Expressing, Irradiation, Transfection, Activity Assay, Staining, Membrane, Gene Expression, Comparison

Journal: bioRxiv

Article Title: TANGO-Light - optogenetic control of transcriptional modulators

doi: 10.1101/2023.05.31.543150

Figure Lengend Snippet: ( A ) Diagram showing the principle where TL is linked to β-Catenin (βCat). ( B ) Relative Firefly luminescence level in HEK293T cells upon photoactivation of TL-βCat for different periods of time. Non transfected cells were used as negative control (NC). ( C ) The mRNA expression of MMP9 , SNAI1 and NANOG genes was determined by qPCR (2–ΔΔCt) 24 after light stimulation of TL-N1CD HEK293T cells. The results are presented as fold change (FC) values (mean□±□SD) normalized to the GAPDH gene expression. All data were analyzed with a one-way ANOVA test and Tukey’s multiple comparisons post-hoc test vs. not light stimulated cells (the time point 0 h and D; dark, respectively). *p□≤□0.05; **p□≤□0.01; ***p□≤□0.001 were considered statistically significant, ns – non significant. All analysed genes showed statistically significant higher expression compared to control cells.

Article Snippet: KOD-Xtreme hot-start DNA polymerase (Merck Millipore), DreamTaqTM Green PCR Master Mix (ThermoFisher Scientific), DpnI restriction enzyme (NEB), Gibson Assembly® Master Mix (NEB), ampicillin (BRAND), DNA Clean & Concentrator and Zyppy Plasmid Kits (Zymoresearch), TurbofectTM Transfection Reagent (ThermoFisher Scientific), Lipofectamine 3000TM (ThermoFisher Scientific),VybrantTM DiD Cell-Labeling Solution (ThermoFisher Scientific), Bright-Glo Luciferase Assay System (Promega), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (PromoCell), plasmids pCDNA3.1(+)-CMV-βarrestin2-TEV (Addgene plasmid #107245; http://n2t.net/addgene:107245 ; RRID:Addgene_107245) and Flag Snai1 6SA (Addgene plasmid #16221; http://n2t.net/addgene:16221 ; RRID:Addgene_16221), plasmid pDONR223_NOTCH1_ICN (Addgene plasmid #82087; http://n2t.net/addgene:82087 ; RRID:Addgene_82087), plasmid GPCR-TANGO and HTLA cells were a gift from the lab of Richard Axel, Howard Hughes Medical Institute, Department of Biochemistry and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, plasmid PA-CXCR4 was a gift from the lab of Minsoo Kim, Ph.D, University of Rochester Medical Center School of Medicine and Dentistry.

Techniques: Transfection, Negative Control, Expressing, Gene Expression, Control

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: Growth factor treatment results in increased SNAI1, increased stemness and decreased let-7 expression. MCF-7, PANC-1 were treated with TGFB1; OVCAR8, OVSAHO were treated with EGF. Levels of control group (cells treated with vehicle control) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression level of SNAI1 ( A ) and of stemness markers (( B ), LIN28A, NANOG, POU5F1 and HMGA2 ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) Left panel: The quantification of number of spheroids per 3000 cells (both first passage and second passage) is shown. Right panel: Phase contrast images of spheroids formed from cells (first passage) as indicated are presented. In each panel, the spheroids formed from control group are presented on the left, those from the treatment group are on the right. Scale bar = 100 μm ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 overexpression results in increased stemness and decreased let-7 expression. Cell lines were transduced with the retroviral expression vector pWZL-Snail or empty vector, pWZL-Empty, in cell lines MCF-7, PANC-1, OVCAR8 and OVSAHO. Levels of control group (cells transduced with pWZL-Empty) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids formed per 3000 cells (both first passage and second passage) as indicated. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Over Expression, Expressing, Transduction, Retroviral, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown reverses stemness and restores let-7 expression. Mesoporous silica nanoparticles coated with hyaluronic acid (HA-MSN) were used to deliver siRNA (siSnail and siControl) in MCF-7, PANC-1, OVCAR8, and OVSAHO. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. Samples were harvested after 24 h (MCF-7, OVCAR8 and OVSAHO) or 72 h (PANC-1). ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids formed per 3000 cells (both first passage and second passage) as indicated. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g , and let-7i ) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Knockdown, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown reduces stemness in patient-derived cells in vitro. HA-MSN were used to deliver siRNA (siSnail and siControl) in PDX cells in vitro. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids per 3000 cells formed from PDX6 in vitro. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g, and let-7i ) expression. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Knockdown, Derivative Assay, In Vitro, Control, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown in vivo reduces stemness gene expression and tumor burden. HA-MSN were used to deliver siRNA (siSnail and siControl) via IV injection to orthotopic PDX in vivo. Tumor samples were harvested and analyzed at necropsy. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ), in tumors. ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and stemness markers LIN28A and HMGA2 ( D ), in tumors. ( E ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i ) expression in tumors. ( F ) Left panel: Representative images of xenograft mice. siControl (upper) and siSnail knockdown (lower). Right panel: Quantitation of bioluminescence at primary sites over six weeks. X axis, days; Y axis, total flux in photons/second relative to day 1. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Knockdown, In Vivo, Gene Expression, IV Injection, Control, Quantitative RT-PCR, Expressing, Western Blot, Quantitation Assay

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 represses let-7 promoters. ( A ) Schematic representation of the promoter region of let- 7 i (upper) and reporter constructs used in luciferase assays (lower diagrams). E1, E2, E3: E-boxes (sequence: CANNTG); MU: Mutated E-boxes; TSS: Transcription start site ( B ) For luciferase assays, HEK293T cells were co-transfected with two plasmids: (1) let-7 promoter luciferase (let-7i, let-7a1/d/f1, let-7a3/b, let-7c) , and (2) either SNAI1 (constitutively expressed, gray bars) or empty vector (black bars). Luminescence activity was measured 48 h thereafter. ( C ) HEK293T cells were co-transfected with either let-7i lucB or let-7i mlucB with or without SNAI1. Luminescence was measured 24 h later. * p < 0.05; ** p < 0.01.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Construct, Luciferase, Sequencing, Transfection, Plasmid Preparation, Activity Assay

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: Growth factor treatment results in increased SNAI1, increased stemness and decreased let-7 expression. MCF-7, PANC-1 were treated with TGFB1; OVCAR8, OVSAHO were treated with EGF. Levels of control group (cells treated with vehicle control) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression level of SNAI1 ( A ) and of stemness markers (( B ), LIN28A, NANOG, POU5F1 and HMGA2 ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) Left panel: The quantification of number of spheroids per 3000 cells (both first passage and second passage) is shown. Right panel: Phase contrast images of spheroids formed from cells (first passage) as indicated are presented. In each panel, the spheroids formed from control group are presented on the left, those from the treatment group are on the right. Scale bar = 100 μm ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Twenty-four hours later PEI reagent was used to transfect cells with 200 ng full length let-7 , truncated let-7i ( lucB ), or mutated let-7i ( mlucB ) promoter luciferase vector in combination with 5 ng Renilla luciferase, and 200 ng SNAI1 -expressing or empty vector (Addgene 16218).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 overexpression results in increased stemness and decreased let-7 expression. Cell lines were transduced with the retroviral expression vector pWZL-Snail or empty vector, pWZL-Empty, in cell lines MCF-7, PANC-1, OVCAR8 and OVSAHO. Levels of control group (cells transduced with pWZL-Empty) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids formed per 3000 cells (both first passage and second passage) as indicated. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Twenty-four hours later PEI reagent was used to transfect cells with 200 ng full length let-7 , truncated let-7i ( lucB ), or mutated let-7i ( mlucB ) promoter luciferase vector in combination with 5 ng Renilla luciferase, and 200 ng SNAI1 -expressing or empty vector (Addgene 16218).

Techniques: Over Expression, Expressing, Transduction, Retroviral, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown reverses stemness and restores let-7 expression. Mesoporous silica nanoparticles coated with hyaluronic acid (HA-MSN) were used to deliver siRNA (siSnail and siControl) in MCF-7, PANC-1, OVCAR8, and OVSAHO. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. Samples were harvested after 24 h (MCF-7, OVCAR8 and OVSAHO) or 72 h (PANC-1). ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids formed per 3000 cells (both first passage and second passage) as indicated. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g , and let-7i ) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Twenty-four hours later PEI reagent was used to transfect cells with 200 ng full length let-7 , truncated let-7i ( lucB ), or mutated let-7i ( mlucB ) promoter luciferase vector in combination with 5 ng Renilla luciferase, and 200 ng SNAI1 -expressing or empty vector (Addgene 16218).

Techniques: Knockdown, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown reduces stemness in patient-derived cells in vitro. HA-MSN were used to deliver siRNA (siSnail and siControl) in PDX cells in vitro. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids per 3000 cells formed from PDX6 in vitro. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g, and let-7i ) expression. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Twenty-four hours later PEI reagent was used to transfect cells with 200 ng full length let-7 , truncated let-7i ( lucB ), or mutated let-7i ( mlucB ) promoter luciferase vector in combination with 5 ng Renilla luciferase, and 200 ng SNAI1 -expressing or empty vector (Addgene 16218).

Techniques: Knockdown, Derivative Assay, In Vitro, Control, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown in vivo reduces stemness gene expression and tumor burden. HA-MSN were used to deliver siRNA (siSnail and siControl) via IV injection to orthotopic PDX in vivo. Tumor samples were harvested and analyzed at necropsy. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ), in tumors. ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and stemness markers LIN28A and HMGA2 ( D ), in tumors. ( E ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i ) expression in tumors. ( F ) Left panel: Representative images of xenograft mice. siControl (upper) and siSnail knockdown (lower). Right panel: Quantitation of bioluminescence at primary sites over six weeks. X axis, days; Y axis, total flux in photons/second relative to day 1. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Twenty-four hours later PEI reagent was used to transfect cells with 200 ng full length let-7 , truncated let-7i ( lucB ), or mutated let-7i ( mlucB ) promoter luciferase vector in combination with 5 ng Renilla luciferase, and 200 ng SNAI1 -expressing or empty vector (Addgene 16218).

Techniques: Knockdown, In Vivo, Gene Expression, IV Injection, Control, Quantitative RT-PCR, Expressing, Western Blot, Quantitation Assay

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 represses let-7 promoters. ( A ) Schematic representation of the promoter region of let- 7 i (upper) and reporter constructs used in luciferase assays (lower diagrams). E1, E2, E3: E-boxes (sequence: CANNTG); MU: Mutated E-boxes; TSS: Transcription start site ( B ) For luciferase assays, HEK293T cells were co-transfected with two plasmids: (1) let-7 promoter luciferase (let-7i, let-7a1/d/f1, let-7a3/b, let-7c) , and (2) either SNAI1 (constitutively expressed, gray bars) or empty vector (black bars). Luminescence activity was measured 48 h thereafter. ( C ) HEK293T cells were co-transfected with either let-7i lucB or let-7i mlucB with or without SNAI1. Luminescence was measured 24 h later. * p < 0.05; ** p < 0.01.

Article Snippet: Twenty-four hours later PEI reagent was used to transfect cells with 200 ng full length let-7 , truncated let-7i ( lucB ), or mutated let-7i ( mlucB ) promoter luciferase vector in combination with 5 ng Renilla luciferase, and 200 ng SNAI1 -expressing or empty vector (Addgene 16218).

Techniques: Construct, Luciferase, Sequencing, Transfection, Plasmid Preparation, Activity Assay