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    Cell Signaling Technology Inc mouse monoclonal anti mitf
    Disruption of <t>MITF–GTF2H1</t> axis represses melanoma growth. a Proliferation of 501 mel cells after transfection with siMITF 1 or three different siGTF2H1 1–3 vs. siSCR. Graphs represent mean ± SD of crystal violet (CV) absorbance from technical triplicates (two-tailed unpaired t -test). Right panels, mRNA expression of MITF and GTF2H1 under RNAi as indicated. b Colony formation assay of transduced 501 mel cells expressing shRNA directed against MITF or GTF2H1 compared to shSCR control. Right panel, bars represent mean ± SD mean of the number of colonies from technical triplicates (two-tailed unpaired t -test). a , b Biological replicates revealed similar results. c End point analysis of tumor growth in shGTF2H1 vs. shSCR expressing melanoma xenografts after subcutaneous transplantation into SCID mice. Tumor growth in shGTF2H1 cohort ( n = 3 of 9 animals) was associated with insufficient repression of GTF2H1 determined by expression analysis in FACS-sorted GFP-positive tumor cells (one-tailed unpaired t -test). Right panel, Kaplan–Meier analysis of tumor-free survival. shGTF2H1 cohort, n = 9 mice; shSCR cohort, n = 10 mice. d Immunohistochemical analysis exhibiting pulmonary spread of genetically modified shGTF2H1 vs. shSCR expressing melanoma cells after tail vein injection. H&E, hematoxylin & eosin staining; MITF, <t>C5</t> <t>monoclonal</t> antibody (red signal). Arrowheads, melanoma cell infiltration. Right panel, Kaplan–Meier analysis of overall survival. shGTF2H1 cohort, n = 5 mice; shSCR cohort, n = 6 mice. P , log-rank test
    Mouse Monoclonal Anti Mitf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Disruption of MITF–GTF2H1 axis represses melanoma growth. a Proliferation of 501 mel cells after transfection with siMITF 1 or three different siGTF2H1 1–3 vs. siSCR. Graphs represent mean ± SD of crystal violet (CV) absorbance from technical triplicates (two-tailed unpaired t -test). Right panels, mRNA expression of MITF and GTF2H1 under RNAi as indicated. b Colony formation assay of transduced 501 mel cells expressing shRNA directed against MITF or GTF2H1 compared to shSCR control. Right panel, bars represent mean ± SD mean of the number of colonies from technical triplicates (two-tailed unpaired t -test). a , b Biological replicates revealed similar results. c End point analysis of tumor growth in shGTF2H1 vs. shSCR expressing melanoma xenografts after subcutaneous transplantation into SCID mice. Tumor growth in shGTF2H1 cohort ( n = 3 of 9 animals) was associated with insufficient repression of GTF2H1 determined by expression analysis in FACS-sorted GFP-positive tumor cells (one-tailed unpaired t -test). Right panel, Kaplan–Meier analysis of tumor-free survival. shGTF2H1 cohort, n = 9 mice; shSCR cohort, n = 10 mice. d Immunohistochemical analysis exhibiting pulmonary spread of genetically modified shGTF2H1 vs. shSCR expressing melanoma cells after tail vein injection. H&E, hematoxylin & eosin staining; MITF, C5 monoclonal antibody (red signal). Arrowheads, melanoma cell infiltration. Right panel, Kaplan–Meier analysis of overall survival. shGTF2H1 cohort, n = 5 mice; shSCR cohort, n = 6 mice. P , log-rank test

    Journal: Oncogene

    Article Title: Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair

    doi: 10.1038/s41388-018-0661-x

    Figure Lengend Snippet: Disruption of MITF–GTF2H1 axis represses melanoma growth. a Proliferation of 501 mel cells after transfection with siMITF 1 or three different siGTF2H1 1–3 vs. siSCR. Graphs represent mean ± SD of crystal violet (CV) absorbance from technical triplicates (two-tailed unpaired t -test). Right panels, mRNA expression of MITF and GTF2H1 under RNAi as indicated. b Colony formation assay of transduced 501 mel cells expressing shRNA directed against MITF or GTF2H1 compared to shSCR control. Right panel, bars represent mean ± SD mean of the number of colonies from technical triplicates (two-tailed unpaired t -test). a , b Biological replicates revealed similar results. c End point analysis of tumor growth in shGTF2H1 vs. shSCR expressing melanoma xenografts after subcutaneous transplantation into SCID mice. Tumor growth in shGTF2H1 cohort ( n = 3 of 9 animals) was associated with insufficient repression of GTF2H1 determined by expression analysis in FACS-sorted GFP-positive tumor cells (one-tailed unpaired t -test). Right panel, Kaplan–Meier analysis of tumor-free survival. shGTF2H1 cohort, n = 9 mice; shSCR cohort, n = 10 mice. d Immunohistochemical analysis exhibiting pulmonary spread of genetically modified shGTF2H1 vs. shSCR expressing melanoma cells after tail vein injection. H&E, hematoxylin & eosin staining; MITF, C5 monoclonal antibody (red signal). Arrowheads, melanoma cell infiltration. Right panel, Kaplan–Meier analysis of overall survival. shGTF2H1 cohort, n = 5 mice; shSCR cohort, n = 6 mice. P , log-rank test

    Article Snippet: For CDK7 promoter analysis we used a mouse monoclonal anti-MITF (Active Motive, 39789) or a rabbit polyclonal anti-c-MYC (Cell Signaling, #9402) and isotype control mouse IgG1 (Biolegend, 401402) and rabbit IgG (Santa Cruz, sc-2027).

    Techniques: Transfection, Two Tailed Test, Expressing, Colony Assay, shRNA, Transplantation Assay, One-tailed Test, Immunohistochemical staining, Genetically Modified, Injection, Staining

    MITF depletion causes loss of MYC and TFIIH kinase. a Phospho-immunoblot analysis of RNA pol II, TFIIH-CAK, and MYC under forced expression of GTF2H1 in 501 mel cells and subsequent siMITF 1 or siSCR transfection compared to EV control. b Transcriptional activity of 501 mel cells as measured by EU incorporation under conditions analogous to a . Graph indicates mean ±SEM of fluorescence intensity in ≥250 nuclei (two-tailed unpaired t -test). c Proliferation of 501 mel cells genetically modified analogous to a . Graphs represent mean ± SD of crystal violet absorbance from technical triplicates (two-tailed unpaired t -test). d Immunoblot analysis of 501 mel cells transfected with siSCR, siMITF 1 , siGTF2H1 1 or siCDK2. e Analysis of CDK7 ubiquitination by immunoprecipitation of transfected FLAG-tagged CDK7 in 501 mel cells treated with 10 µM MG132 or 1 µM bortezomib (BZ) compared to DMSO control. Left panel, ubiquitination input control; right panel, FLAG-directed immunoprecipitation of ubiquitinated CDK7 using anti-ubiquitin antibody. Detection of CDK7-FLAG with anti-FLAG antibody as loading control. f Immunoblot analysis of 501 mel cells transfected with siSCR, siMITF 1 or siMYC under proteasome inhibition with 10 μM MG132 vs. DMSO control. g Immunoblot analysis of CDK7 expression under MITF depletion vs. control in 501 mel cells upon expression of c-MYC wild type, MYC box I and II, and transcription- and dimerization-deficient mutants of c-MYC and MITF. Leucine zipper was deleted to prevent dominant-negative effects on wild-type MAX or MITF by sequestration. Upper arrowhead: MYC WT, MYC AADA ΔLZ and MITF ADAA ΔLZ; lower arrowhead: MYC I + II. Actin used as loading control. a – g Experiments were performed twice independently with very similar results

    Journal: Oncogene

    Article Title: Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair

    doi: 10.1038/s41388-018-0661-x

    Figure Lengend Snippet: MITF depletion causes loss of MYC and TFIIH kinase. a Phospho-immunoblot analysis of RNA pol II, TFIIH-CAK, and MYC under forced expression of GTF2H1 in 501 mel cells and subsequent siMITF 1 or siSCR transfection compared to EV control. b Transcriptional activity of 501 mel cells as measured by EU incorporation under conditions analogous to a . Graph indicates mean ±SEM of fluorescence intensity in ≥250 nuclei (two-tailed unpaired t -test). c Proliferation of 501 mel cells genetically modified analogous to a . Graphs represent mean ± SD of crystal violet absorbance from technical triplicates (two-tailed unpaired t -test). d Immunoblot analysis of 501 mel cells transfected with siSCR, siMITF 1 , siGTF2H1 1 or siCDK2. e Analysis of CDK7 ubiquitination by immunoprecipitation of transfected FLAG-tagged CDK7 in 501 mel cells treated with 10 µM MG132 or 1 µM bortezomib (BZ) compared to DMSO control. Left panel, ubiquitination input control; right panel, FLAG-directed immunoprecipitation of ubiquitinated CDK7 using anti-ubiquitin antibody. Detection of CDK7-FLAG with anti-FLAG antibody as loading control. f Immunoblot analysis of 501 mel cells transfected with siSCR, siMITF 1 or siMYC under proteasome inhibition with 10 μM MG132 vs. DMSO control. g Immunoblot analysis of CDK7 expression under MITF depletion vs. control in 501 mel cells upon expression of c-MYC wild type, MYC box I and II, and transcription- and dimerization-deficient mutants of c-MYC and MITF. Leucine zipper was deleted to prevent dominant-negative effects on wild-type MAX or MITF by sequestration. Upper arrowhead: MYC WT, MYC AADA ΔLZ and MITF ADAA ΔLZ; lower arrowhead: MYC I + II. Actin used as loading control. a – g Experiments were performed twice independently with very similar results

    Article Snippet: For CDK7 promoter analysis we used a mouse monoclonal anti-MITF (Active Motive, 39789) or a rabbit polyclonal anti-c-MYC (Cell Signaling, #9402) and isotype control mouse IgG1 (Biolegend, 401402) and rabbit IgG (Santa Cruz, sc-2027).

    Techniques: Western Blot, Expressing, Transfection, Activity Assay, Fluorescence, Two Tailed Test, Genetically Modified, Immunoprecipitation, Inhibition, Dominant Negative Mutation

    c-MYC rescues general transcription and prevents senescence in the absence of MITF. a Immunoblot analysis of 501 mel cells under retrovirus-driven c-MYC expression compared to EV and subsequent siMITF 1 vs. siSCR transfection. b MITF, c-MYC, and CDK7 mRNA expression in 501 mel cells under conditions analogous to a . Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t -test). c Transcriptional activity measured as EU incorporation in 501 mel cells in analogy to a and b . Graphs indicate mean ±SEM of fluorescence intensity in ≥250 nuclei (two-tailed unpaired t -test). d Proliferation of 501 mel cells in analogy to a – c . Graphs represent mean ± SD of crystal violet absorbance from technical triplicates (two-tailed unpaired t -test). e SA-β-gal positivity in 501 mel cells at day 3 in analogy to d . Data represent mean ± SD from technical triplicates (two-tailed unpaired t -test). Micrographs display SA-β-gal signals and Hoechst 33342 nuclear staining under corresponding conditions (scale: 50 µm). f Regression analysis of MITF and MYC single-cell mRNA expression from a panel of metastatic melanomas (GSE72056). g ChIP‐seq tracks of MITF-binding signals at FUBP2/KHSRP gene locus in primary melanocytes, and 501 mel and COLO829 BT168F melanoma cell lines. Green arrowheads indicate MITF-binding consensus sequences (E box) at the promoter and first intronic region of the FUBP2/KHSRP gene. GEO accession numbers are listed under Materials and Methods. h MITF, c-MYC, and FUBP2 mRNA expression in 501 mel cells after siSCR, siMITF 1 , or siFUBP2 transfection. Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t -test). i Immunofluorescence and immunoblot analyses of MITF, MYC, and FUBP2 protein expression in analogy to h . DRAQ5 used for nuclear staining and actin used as loading control (two-tailed unpaired t -test of c-MYC fluorescence intensity in 100 nuclei is indicated). a – e , h , i Experiments were repeated twice with comparable results

    Journal: Oncogene

    Article Title: Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair

    doi: 10.1038/s41388-018-0661-x

    Figure Lengend Snippet: c-MYC rescues general transcription and prevents senescence in the absence of MITF. a Immunoblot analysis of 501 mel cells under retrovirus-driven c-MYC expression compared to EV and subsequent siMITF 1 vs. siSCR transfection. b MITF, c-MYC, and CDK7 mRNA expression in 501 mel cells under conditions analogous to a . Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t -test). c Transcriptional activity measured as EU incorporation in 501 mel cells in analogy to a and b . Graphs indicate mean ±SEM of fluorescence intensity in ≥250 nuclei (two-tailed unpaired t -test). d Proliferation of 501 mel cells in analogy to a – c . Graphs represent mean ± SD of crystal violet absorbance from technical triplicates (two-tailed unpaired t -test). e SA-β-gal positivity in 501 mel cells at day 3 in analogy to d . Data represent mean ± SD from technical triplicates (two-tailed unpaired t -test). Micrographs display SA-β-gal signals and Hoechst 33342 nuclear staining under corresponding conditions (scale: 50 µm). f Regression analysis of MITF and MYC single-cell mRNA expression from a panel of metastatic melanomas (GSE72056). g ChIP‐seq tracks of MITF-binding signals at FUBP2/KHSRP gene locus in primary melanocytes, and 501 mel and COLO829 BT168F melanoma cell lines. Green arrowheads indicate MITF-binding consensus sequences (E box) at the promoter and first intronic region of the FUBP2/KHSRP gene. GEO accession numbers are listed under Materials and Methods. h MITF, c-MYC, and FUBP2 mRNA expression in 501 mel cells after siSCR, siMITF 1 , or siFUBP2 transfection. Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t -test). i Immunofluorescence and immunoblot analyses of MITF, MYC, and FUBP2 protein expression in analogy to h . DRAQ5 used for nuclear staining and actin used as loading control (two-tailed unpaired t -test of c-MYC fluorescence intensity in 100 nuclei is indicated). a – e , h , i Experiments were repeated twice with comparable results

    Article Snippet: For CDK7 promoter analysis we used a mouse monoclonal anti-MITF (Active Motive, 39789) or a rabbit polyclonal anti-c-MYC (Cell Signaling, #9402) and isotype control mouse IgG1 (Biolegend, 401402) and rabbit IgG (Santa Cruz, sc-2027).

    Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Two Tailed Test, Activity Assay, Fluorescence, Staining, ChIP-sequencing, Binding Assay, Immunofluorescence

    CDK7 is a direct transcriptional target of MITF and c-MYC. a Time course of CDK7 mRNA expression in 501 mel cells transfected with siMITF 1 vs. control siRNA under treatment with phorbol ester (TPA). Data represent mean ± SD from technical triplicates (two-tailed paired t -test). b Immunoblot analysis of MITF, c-MYC and CDK7 protein expression in 501 mel cells under TPA treatment. Lower and upper arrowheads indicate non-phosphorylated (54 kd) and phosphorylated (60 kd) MITF forms, respectively. Actin used as loading control. c ChIP-seq tracks of MITF (marked red) and c-MYC (marked blue) binding signals at CDK7 gene locus in primary melanocytes and primary melanomas, melanoma cell lines and non-melanocytic cancer cells, respectively. Green arrowhead indicates E box consensus sequence −86 base pairs upstream of the transcriptional start site of CDK7. GEO accession numbers of corresponding data sets are listed under Materials and methods. d ChIP, in vivo occupancy of MITF at CDK7 promoter in 501 mel cells in the presence or absence of forced c-MYC expression (OE overexpression) compared to empty vector (EV) including intron sequence and IgG controls. Mean ± SD from technical triplicates. e ChIP, in vivo occupancy of exogenous c-MYC (OE) at CDK7 promoter in 501 mel cells under siSCR or siMITF 1 transfection. Intron sequence and IgG were used as controls. Mean ± SD is presented from technical triplicates. d , e Two-tailed paired t -test. a , b , d , e Experiments were performed twice and biological replicates revealed very similar results

    Journal: Oncogene

    Article Title: Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair

    doi: 10.1038/s41388-018-0661-x

    Figure Lengend Snippet: CDK7 is a direct transcriptional target of MITF and c-MYC. a Time course of CDK7 mRNA expression in 501 mel cells transfected with siMITF 1 vs. control siRNA under treatment with phorbol ester (TPA). Data represent mean ± SD from technical triplicates (two-tailed paired t -test). b Immunoblot analysis of MITF, c-MYC and CDK7 protein expression in 501 mel cells under TPA treatment. Lower and upper arrowheads indicate non-phosphorylated (54 kd) and phosphorylated (60 kd) MITF forms, respectively. Actin used as loading control. c ChIP-seq tracks of MITF (marked red) and c-MYC (marked blue) binding signals at CDK7 gene locus in primary melanocytes and primary melanomas, melanoma cell lines and non-melanocytic cancer cells, respectively. Green arrowhead indicates E box consensus sequence −86 base pairs upstream of the transcriptional start site of CDK7. GEO accession numbers of corresponding data sets are listed under Materials and methods. d ChIP, in vivo occupancy of MITF at CDK7 promoter in 501 mel cells in the presence or absence of forced c-MYC expression (OE overexpression) compared to empty vector (EV) including intron sequence and IgG controls. Mean ± SD from technical triplicates. e ChIP, in vivo occupancy of exogenous c-MYC (OE) at CDK7 promoter in 501 mel cells under siSCR or siMITF 1 transfection. Intron sequence and IgG were used as controls. Mean ± SD is presented from technical triplicates. d , e Two-tailed paired t -test. a , b , d , e Experiments were performed twice and biological replicates revealed very similar results

    Article Snippet: For CDK7 promoter analysis we used a mouse monoclonal anti-MITF (Active Motive, 39789) or a rabbit polyclonal anti-c-MYC (Cell Signaling, #9402) and isotype control mouse IgG1 (Biolegend, 401402) and rabbit IgG (Santa Cruz, sc-2027).

    Techniques: Expressing, Transfection, Two Tailed Test, Western Blot, ChIP-sequencing, Binding Assay, Sequencing, In Vivo, Over Expression, Plasmid Preparation

    Model of TFIIH-CAK regulation by MITF and c-MYC. In the melanocytic lineage, the microphthalmia-associated transcription factor (MITF) determines transcriptional homeostasis and genomic integrity through regulation of the transcription factor II H (TFIIH) and the CDK-activating kinase (CAK) complex. (I) MITF controls general transcription and UVR-induced nucleotide excision repair by targeting the transcription initiation complex through direct transactivation of GTF2H1 as a core element of TFIIH. (II) The melanocyte master regulator MITF and its structural homolog c-MYC control the expression of CDK7, the catalytic subunit of CAK complex, which has a dual role in transcription and cell cycle activities. (III) MITF controls MYC activities by transactivation of fuse binding protein (FUBP2) involved in pulse regulation of MYC. In addition, MYC is likely affected by MITF-dependent effects on proliferation

    Journal: Oncogene

    Article Title: Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair

    doi: 10.1038/s41388-018-0661-x

    Figure Lengend Snippet: Model of TFIIH-CAK regulation by MITF and c-MYC. In the melanocytic lineage, the microphthalmia-associated transcription factor (MITF) determines transcriptional homeostasis and genomic integrity through regulation of the transcription factor II H (TFIIH) and the CDK-activating kinase (CAK) complex. (I) MITF controls general transcription and UVR-induced nucleotide excision repair by targeting the transcription initiation complex through direct transactivation of GTF2H1 as a core element of TFIIH. (II) The melanocyte master regulator MITF and its structural homolog c-MYC control the expression of CDK7, the catalytic subunit of CAK complex, which has a dual role in transcription and cell cycle activities. (III) MITF controls MYC activities by transactivation of fuse binding protein (FUBP2) involved in pulse regulation of MYC. In addition, MYC is likely affected by MITF-dependent effects on proliferation

    Article Snippet: For CDK7 promoter analysis we used a mouse monoclonal anti-MITF (Active Motive, 39789) or a rabbit polyclonal anti-c-MYC (Cell Signaling, #9402) and isotype control mouse IgG1 (Biolegend, 401402) and rabbit IgG (Santa Cruz, sc-2027).

    Techniques: Expressing, Binding Assay