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-028005 sureprint g3 mouse ge 8x60k microarray (Agilent technologies)

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Agilent technologies -028005 sureprint g3 mouse ge 8x60k microarray

WT BMDMs were treated with IFNβ (100 U/mL), zVAD-fmk (50 µM), and Nec-1 (10 µM). At 6 h, differential gene expression and GSEA were performed on <t>microarray</t> data comparing WT BMDMs treated with IFNβ, IFNβ + zVAD, and IFNβ + zVAD+Nec1 ( A – D ). Expression of TNFα was measured in the supernatants collected at 7 h after treating the WT BMDMs with EMR (10 µM) and different LPS concentrations ( E ). Expression of IFNβ, and IL-12p70 was measured in the supernatants collected at 7 h after treating the WT BMDMs with LPS (1 ng/mL) and EMR (10 µM) at 7 h ( F , G ). H , I WT BMDMs were stimulated with LPS (1 ng/mL), EMR (10 µM), Nec1-s (10 µM) and the impact on cell death was evaluated at 24 h by MTT ( H ). Secretion of TNFα was measured in supernatants collected at 7 h ( I ), and the activation of various proteins was evaluated by performing western blotting of cell extracts collected at various time intervals ( J ). Each experiment was repeated at least three times. (** P < 0.01, *** P < 0.001, **** P < 0.0001).

028005 Sureprint G3 Mouse Ge 8x60k Microarray, supplied by Agilent technologies, 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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1) Product Images from "Regulation of Zfp36 by ISGF3 and MK2 restricts the expression of inflammatory cytokines during necroptosis stimulation"

Article Title: Regulation of Zfp36 by ISGF3 and MK2 restricts the expression of inflammatory cytokines during necroptosis stimulation

Journal: Cell Death & Disease

doi: 10.1038/s41419-024-06964-4

WT BMDMs were treated with IFNβ (100 U/mL), zVAD-fmk (50 µM), and Nec-1 (10 µM). At 6 h, differential gene expression and GSEA were performed on microarray data comparing WT BMDMs treated with IFNβ, IFNβ + zVAD, and IFNβ + zVAD+Nec1 ( A – D ). Expression of TNFα was measured in the supernatants collected at 7 h after treating the WT BMDMs with EMR (10 µM) and different LPS concentrations ( E ). Expression of IFNβ, and IL-12p70 was measured in the supernatants collected at 7 h after treating the WT BMDMs with LPS (1 ng/mL) and EMR (10 µM) at 7 h ( F , G ). H , I WT BMDMs were stimulated with LPS (1 ng/mL), EMR (10 µM), Nec1-s (10 µM) and the impact on cell death was evaluated at 24 h by MTT ( H ). Secretion of TNFα was measured in supernatants collected at 7 h ( I ), and the activation of various proteins was evaluated by performing western blotting of cell extracts collected at various time intervals ( J ). Each experiment was repeated at least three times. (** P < 0.01, *** P < 0.001, **** P < 0.0001).

Figure Legend Snippet: WT BMDMs were treated with IFNβ (100 U/mL), zVAD-fmk (50 µM), and Nec-1 (10 µM). At 6 h, differential gene expression and GSEA were performed on microarray data comparing WT BMDMs treated with IFNβ, IFNβ + zVAD, and IFNβ + zVAD+Nec1 ( A – D ). Expression of TNFα was measured in the supernatants collected at 7 h after treating the WT BMDMs with EMR (10 µM) and different LPS concentrations ( E ). Expression of IFNβ, and IL-12p70 was measured in the supernatants collected at 7 h after treating the WT BMDMs with LPS (1 ng/mL) and EMR (10 µM) at 7 h ( F , G ). H , I WT BMDMs were stimulated with LPS (1 ng/mL), EMR (10 µM), Nec1-s (10 µM) and the impact on cell death was evaluated at 24 h by MTT ( H ). Secretion of TNFα was measured in supernatants collected at 7 h ( I ), and the activation of various proteins was evaluated by performing western blotting of cell extracts collected at various time intervals ( J ). Each experiment was repeated at least three times. (** P < 0.01, *** P < 0.001, **** P < 0.0001).

Techniques Used: Expressing, Microarray, Activation Assay, Western Blot

WT and Ifnar1 −/− BMDMs were treated with LPS (1 ng/mL) in the presence of zVAD-fmk (50 µM) for 6 h and differential gene expression and GSEA was performed on microarray data comparing WT BMDMs treated with LPS+zVAD with Ifnar1 −/− BMDMs treated with LPS+zVAD ( A – D ). E Expression levels of TNFα, IFNβ, IL-6, and IL-10 were measured in the supernatants collected after treating WT and Ifnar1 −/− BMDMs for 7 h with LPS (1 ng/mL) and EMR (10 µM). F , G Western blot analysis was performed in cell extracts collected from WT and Ifnar1 −/− BMDMs at different time intervals following treatment with LPS (1 ng/mL) and EMR (10 µM). Each experiment was repeated at least three times. (**** P < 0.0001).

Figure Legend Snippet: WT and Ifnar1 −/− BMDMs were treated with LPS (1 ng/mL) in the presence of zVAD-fmk (50 µM) for 6 h and differential gene expression and GSEA was performed on microarray data comparing WT BMDMs treated with LPS+zVAD with Ifnar1 −/− BMDMs treated with LPS+zVAD ( A – D ). E Expression levels of TNFα, IFNβ, IL-6, and IL-10 were measured in the supernatants collected after treating WT and Ifnar1 −/− BMDMs for 7 h with LPS (1 ng/mL) and EMR (10 µM). F , G Western blot analysis was performed in cell extracts collected from WT and Ifnar1 −/− BMDMs at different time intervals following treatment with LPS (1 ng/mL) and EMR (10 µM). Each experiment was repeated at least three times. (**** P < 0.0001).

Techniques Used: Expressing, Microarray, Western Blot

other:

Article Title: Transcriptomic profiles of tissues from rats treated with anticancer drug combinations
Article Snippet: GSE84048 , 28011623 , 2017 , Mouse , Sunitinib , NA , Agilent-028005 SurePrint G3 Mouse GE 8 × 60 K Microarray.

Article Title: Identification of Immune Infiltration and Effective Immune Biomarkers in Acute Lung Injury by Bioinformatics Analysis
Article Snippet: GSE104214 , Agilent-028005 SurePrint G3 Mouse GE 8 × 60 K Microarray , 11 , 6.

Article Title: Maternal α-casein deficiency extends the lifespan of offspring and programmes their body composition.
Article Snippet: For microarray (Agilent-028005 Whole Genome Mouse 8 × 60 k Array, Agilent Technologies), samples (n = 4) derived from liver of mice at day 21 (at the point of weaning) and day 100 from offspring nursed by α-casein deficient dams or wild-type dams (4 experimental groups in total); 100 ng of total RNA was labelled using One Colour Low Input Quick Amp Labelling kit (5190–2305, Agilent Technologies).

Article Title: The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-ĸB subunits
Article Snippet: RNA quantification and purity were measured using the Nanodrop ND1000 spectrophotometer (Thermo Scientific), with samples submitted for microarray profiling (Beckman Coulter Genomics, Morrisville, NC).

Article Title: Regulation of Zfp36 by ISGF3 and MK2 restricts the expression of inflammatory cytokines during necroptosis stimulation
Article Snippet: Labeled cRNA was hybridized to Agilent-028005 SurePrint G3 Mouse GE 8x60K Microarray (GPL10787).

Article Title: Miz1 Deficiency in the Mammary Gland Causes a Lactation Defect by Attenuated Stat5 Expression and Phosphorylation
Article Snippet: A mouse genome Agilent-028005 array was used for the analysis of gene expression of 1 st pregnancy lactation day 6 in vivo samples (n = 4 per genotype).

Labeling:

Article Title: Toll-Like Receptors Drive Specific Patterns of Tolerance and Training on Restimulation of Macrophages
Article Snippet: .. Labeled RNA (cRNA) was hybridized to Agilent mouse 8 × 60 K microarrays (Agilent-028005). ..

Image Search Results

Journal: Cell Death & Disease

Article Title: Regulation of Zfp36 by ISGF3 and MK2 restricts the expression of inflammatory cytokines during necroptosis stimulation

doi: 10.1038/s41419-024-06964-4

Figure Lengend Snippet: WT BMDMs were treated with IFNβ (100 U/mL), zVAD-fmk (50 µM), and Nec-1 (10 µM). At 6 h, differential gene expression and GSEA were performed on microarray data comparing WT BMDMs treated with IFNβ, IFNβ + zVAD, and IFNβ + zVAD+Nec1 ( A – D ). Expression of TNFα was measured in the supernatants collected at 7 h after treating the WT BMDMs with EMR (10 µM) and different LPS concentrations ( E ). Expression of IFNβ, and IL-12p70 was measured in the supernatants collected at 7 h after treating the WT BMDMs with LPS (1 ng/mL) and EMR (10 µM) at 7 h ( F , G ). H , I WT BMDMs were stimulated with LPS (1 ng/mL), EMR (10 µM), Nec1-s (10 µM) and the impact on cell death was evaluated at 24 h by MTT ( H ). Secretion of TNFα was measured in supernatants collected at 7 h ( I ), and the activation of various proteins was evaluated by performing western blotting of cell extracts collected at various time intervals ( J ). Each experiment was repeated at least three times. (** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Labeled cRNA was hybridized to Agilent-028005 SurePrint G3 Mouse GE 8x60K Microarray (GPL10787).

Techniques: Expressing, Microarray, Activation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Regulation of Zfp36 by ISGF3 and MK2 restricts the expression of inflammatory cytokines during necroptosis stimulation

doi: 10.1038/s41419-024-06964-4

Figure Lengend Snippet: WT and Ifnar1 −/− BMDMs were treated with LPS (1 ng/mL) in the presence of zVAD-fmk (50 µM) for 6 h and differential gene expression and GSEA was performed on microarray data comparing WT BMDMs treated with LPS+zVAD with Ifnar1 −/− BMDMs treated with LPS+zVAD ( A – D ). E Expression levels of TNFα, IFNβ, IL-6, and IL-10 were measured in the supernatants collected after treating WT and Ifnar1 −/− BMDMs for 7 h with LPS (1 ng/mL) and EMR (10 µM). F , G Western blot analysis was performed in cell extracts collected from WT and Ifnar1 −/− BMDMs at different time intervals following treatment with LPS (1 ng/mL) and EMR (10 µM). Each experiment was repeated at least three times. (**** P < 0.0001).

Article Snippet: Labeled cRNA was hybridized to Agilent-028005 SurePrint G3 Mouse GE 8x60K Microarray (GPL10787).

Techniques: Expressing, Microarray, Western Blot

( A ) The efficiency of exogenous DiPRO1 overexpression was verified by mRNA expression analysis. The qRT-PCR results were normalized to the GAPDH gene and represent three independent total RNA extractions measured in triplicate, FC ± SD. Ct = 45 was taken for exDiPRO1 ORF expression in control cells. The P value indicates the difference between endogenous (enDiPRO1) and exogenous (enDiPRO1) DiPRO1 expression, n = 3. ( B ) Western blotting revealed exogenous expression of DiPRO1 protein. Immunoblotting assays were performed on whole-cell extracts, with DiPRO1 protein labeled with the HA-tag. Actin was used as an internal control. ( C ) Morphological changes in myoblasts stably expressing pDiPRO1. Real images (left panel) and cells stained in MGG reagent (middle and right panels) are shown.The red arrows point to a magnified view of an individual cell. Scale bar: 50 µm (left, middle) and 10 µm (right). ( D ) DiPRO1 overexpression induces changes in cell dimension. L: cell length, W: cell width, Sc: cytoplasmic surface, Sn: nuclear surface, N/C: nuclear-cytoplasmic ratio. Unit of length = µm. Results represent m ± SD, n = 20. T -test was used for statistical analysis. ( E ) DiPRO1 induces myoblast proliferation. The proliferation rate was estimated for DiPRO1-overexpressing myoblasts (Myo_pDiPRO1) and their control (Myo_Ctl) and compared with RMS cells (RMS_Ctl) by Counting Kit 8 (Sigma-Aldrich). Cell number was determined using a titration curve performed at different cell dilutions from 0 to 25,000 cells/well. Cells were then seeded at 1.0 × 10 4 , 1.5 × 10 4 , and 2.5 × 10 4 cells/well and absorbance was measured at 460 nm after 72 h of proliferation. Viable cells were compared to the initial number of cells. Data were expressed as mean % relative to initial cell number ± SD and represent three independent experiments, t -test, n = 3. ( F ) Overexpression of DiPRO1 results in the block of myoblast differentiation. Cells were stained with anti-tropomyosin (green), anti-actin (red) and DAPI (blue) antibodies. The merge contains the combined images of three different stainings. Scale bar 120 µm. ( G ) DiPRO1 mRNA inhibition was verified by RT-qPCR analysis. Data were normalized to GAPDH expression and DiPRO1 expression in control cells was set to 100%, proportions (%) ± SD, n = 3 corresponding to three independent experiments measured twice, t -test. ( H ) Cell cycle analysis of DiPRO1-depleted myoblasts was performed one week after transduction. Transduced myoblasts were stained with propidium iodide (PI). The percentage of dead and viable cells in each phase, according to DNA content, was determined by flow cytometry and compared with control cells, proportions (%) ± SD, n = 3 corresponding to three independent experiments. ( I ) Morphological changes in myoblasts resulting from DiPRO1 knockdown (shDiPRO1) one week after transduction (top). Transduction efficiency was verified by confocal microscopy using the shDiPRO1 vector expressing GFP (top left). Scale bar 100 µm. Length (L) and width (W) of cells were measured (bottom). Length unit = µm. Results are presented as m ± SD, number of fields n = 20. ( J ) DiPRO1 KD contributes to myogenic gene expression. Results of RT-qPCR analysis were normalized to GAPDH expression and presented as ratios to shCtl. The t -test was applied for statistical difference with the appropriate control, FC ± SD, n = 3, indicating three independent experiments performed in duplicate. ( K ) GO terms of significantly regulated biological processes are summarized using the genetic interaction network. Cytoscape software with ClueGO plug-in was used, P < 0.01, kappa score threshold 0.4, n = 6–7. ( L ) Gene signatures of neuromuscular development, and cell cycle and proliferation pathways correlated with DiPRO1 expression versus control, n = 6–7. ( M ) Endogenous DiPRO1 gene expression in RMS, proliferating (Myo-prolif) and differentiating (Myo-dif) myoblasts. The qRT-PCR results were normalized to the GAPDH gene and represent three independent total RNA extractions measured twice, m ± SD, n = 3, t -test. Data information: In ( A – G ), two myoblast cell lines were transduced with a retroviral vector expressing the DiPRO1 ORF (pDiPRO1) and compared with parental control counterparts (Ctl). In ( G – J ), two human myoblast cell lines were transduced with a lentivirus vector expressing a shRNA targeting the DiPRO1 gene (shDiPRO1) and compared with myoblasts transduced with the equivalent vector expressing a nontargeting shRNA (shCTL). In ( K , L ), global transcriptome analysis by microarray was implemented using total mRNA extracted from myoblast cell lines: with DiPRO1 knockdown (shDiPRO1) or overexpression (pDiPRO1) and corresponding controls. Three or four separate extractions were performed from two cell lines ( n = 6–7 per condition). Differentially expressed genes in pDiPRO1- and shDiPRO1-myoblasts compared with corresponding controls ( P < 0.05). The Limma R package was used for statistical analysis. In ( A , D , E , G – J , M ), *, **, and *** indicate significant differences from the corresponding control, P < 0.05, 0.01, and 0.001, respectively. .

Journal: EMBO Molecular Medicine

Article Title: DiPRO1 distinctly reprograms muscle and mesenchymal cancer cells

doi: 10.1038/s44321-024-00097-z

Figure Lengend Snippet: ( A ) The efficiency of exogenous DiPRO1 overexpression was verified by mRNA expression analysis. The qRT-PCR results were normalized to the GAPDH gene and represent three independent total RNA extractions measured in triplicate, FC ± SD. Ct = 45 was taken for exDiPRO1 ORF expression in control cells. The P value indicates the difference between endogenous (enDiPRO1) and exogenous (enDiPRO1) DiPRO1 expression, n = 3. ( B ) Western blotting revealed exogenous expression of DiPRO1 protein. Immunoblotting assays were performed on whole-cell extracts, with DiPRO1 protein labeled with the HA-tag. Actin was used as an internal control. ( C ) Morphological changes in myoblasts stably expressing pDiPRO1. Real images (left panel) and cells stained in MGG reagent (middle and right panels) are shown.The red arrows point to a magnified view of an individual cell. Scale bar: 50 µm (left, middle) and 10 µm (right). ( D ) DiPRO1 overexpression induces changes in cell dimension. L: cell length, W: cell width, Sc: cytoplasmic surface, Sn: nuclear surface, N/C: nuclear-cytoplasmic ratio. Unit of length = µm. Results represent m ± SD, n = 20. T -test was used for statistical analysis. ( E ) DiPRO1 induces myoblast proliferation. The proliferation rate was estimated for DiPRO1-overexpressing myoblasts (Myo_pDiPRO1) and their control (Myo_Ctl) and compared with RMS cells (RMS_Ctl) by Counting Kit 8 (Sigma-Aldrich). Cell number was determined using a titration curve performed at different cell dilutions from 0 to 25,000 cells/well. Cells were then seeded at 1.0 × 10 4 , 1.5 × 10 4 , and 2.5 × 10 4 cells/well and absorbance was measured at 460 nm after 72 h of proliferation. Viable cells were compared to the initial number of cells. Data were expressed as mean % relative to initial cell number ± SD and represent three independent experiments, t -test, n = 3. ( F ) Overexpression of DiPRO1 results in the block of myoblast differentiation. Cells were stained with anti-tropomyosin (green), anti-actin (red) and DAPI (blue) antibodies. The merge contains the combined images of three different stainings. Scale bar 120 µm. ( G ) DiPRO1 mRNA inhibition was verified by RT-qPCR analysis. Data were normalized to GAPDH expression and DiPRO1 expression in control cells was set to 100%, proportions (%) ± SD, n = 3 corresponding to three independent experiments measured twice, t -test. ( H ) Cell cycle analysis of DiPRO1-depleted myoblasts was performed one week after transduction. Transduced myoblasts were stained with propidium iodide (PI). The percentage of dead and viable cells in each phase, according to DNA content, was determined by flow cytometry and compared with control cells, proportions (%) ± SD, n = 3 corresponding to three independent experiments. ( I ) Morphological changes in myoblasts resulting from DiPRO1 knockdown (shDiPRO1) one week after transduction (top). Transduction efficiency was verified by confocal microscopy using the shDiPRO1 vector expressing GFP (top left). Scale bar 100 µm. Length (L) and width (W) of cells were measured (bottom). Length unit = µm. Results are presented as m ± SD, number of fields n = 20. ( J ) DiPRO1 KD contributes to myogenic gene expression. Results of RT-qPCR analysis were normalized to GAPDH expression and presented as ratios to shCtl. The t -test was applied for statistical difference with the appropriate control, FC ± SD, n = 3, indicating three independent experiments performed in duplicate. ( K ) GO terms of significantly regulated biological processes are summarized using the genetic interaction network. Cytoscape software with ClueGO plug-in was used, P < 0.01, kappa score threshold 0.4, n = 6–7. ( L ) Gene signatures of neuromuscular development, and cell cycle and proliferation pathways correlated with DiPRO1 expression versus control, n = 6–7. ( M ) Endogenous DiPRO1 gene expression in RMS, proliferating (Myo-prolif) and differentiating (Myo-dif) myoblasts. The qRT-PCR results were normalized to the GAPDH gene and represent three independent total RNA extractions measured twice, m ± SD, n = 3, t -test. Data information: In ( A – G ), two myoblast cell lines were transduced with a retroviral vector expressing the DiPRO1 ORF (pDiPRO1) and compared with parental control counterparts (Ctl). In ( G – J ), two human myoblast cell lines were transduced with a lentivirus vector expressing a shRNA targeting the DiPRO1 gene (shDiPRO1) and compared with myoblasts transduced with the equivalent vector expressing a nontargeting shRNA (shCTL). In ( K , L ), global transcriptome analysis by microarray was implemented using total mRNA extracted from myoblast cell lines: with DiPRO1 knockdown (shDiPRO1) or overexpression (pDiPRO1) and corresponding controls. Three or four separate extractions were performed from two cell lines ( n = 6–7 per condition). Differentially expressed genes in pDiPRO1- and shDiPRO1-myoblasts compared with corresponding controls ( P < 0.05). The Limma R package was used for statistical analysis. In ( A , D , E , G – J , M ), *, **, and *** indicate significant differences from the corresponding control, P < 0.05, 0.01, and 0.001, respectively. .

Article Snippet: Gene expression analysis was performed using an Agilent® SurePrint G3 Human GE 8x60K Microarray (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Single Color Labeling Kit (Low Input Quick Amp Labeling Kit 034949) adapted for small amounts of total RNA (100 ng total RNA per reaction).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control, Western Blot, Labeling, Stable Transfection, Staining, Titration, Blocking Assay, Inhibition, Cell Cycle Assay, Transduction, Flow Cytometry, Knockdown, Confocal Microscopy, Plasmid Preparation, Software, Retroviral, shRNA, Microarray

Journal: EMBO Molecular Medicine

Article Title: DiPRO1 distinctly reprograms muscle and mesenchymal cancer cells

doi: 10.1038/s44321-024-00097-z

Figure Lengend Snippet: ( A – C ) Myoblasts were stably transduced with a retroviral vector expressing the DiPRO1 ORF (pDiPRO1, n = 6) and compared with their parental counterparts (Ctl, n = 6). ( A ) Heatmap revealing differential expression of a set of genes in control (blue) and pDiPRO1 (red) samples. Upregulation of DiPRO1 gene expression ( B ) stimulates expression of PAX-family genes ( C ). ( D ) Cell death analysis of shDiPRO1 myoblasts. The cells transduced with lentiviral vector expressing shDiPRO1 or nontargeting shCtl, were stained with propidium iodide (PI) and analyzed by flow cytometry. The percentage of dead cells according to DNA content was compared to control cells, proportions (%) ± SD, n = 3 corresponding to three independent experiments. ( E – G ) Human myoblasts were transduced with a lentiviral vector expressing a DiPRO1-targeting shRNA (shDiPRO1, n = 7) and compared to myoblasts transduced with the equivalent vector expressing a nontargeting shRNA (shCTL, n = 6). The analysis was performed one week post-transduction. ( E ) Heatmap revealing changes in gene expression profile related to DiPRO1 knockdown in myoblasts. ( F ) DiPRO1 expression was significantly inhibited by shDiPRO1 (red) relative to shCtl (blue). ( G ) Clustered functional network of 185 upregulated (UP) and 136 downregulated (DW) genes encompassing muscle-related functions, negatively correlated with DiPRO1 expression in myoblats. Muscle GO terms are represented by nodes. Two networks were confronted. Green clusters correspond to upregulated myogenic genes, while lilac clusters correspond to downregulated genes in shDiPRO1 versus shCtl. Cytoscape software with ClueGO plug-in was used, kappa score = 0.4. ( H ) PCA analysis of differentially expressed genes separates pDiPRO1 ( n = 6) and control according to the first principal component and shDiPRO1 ( n = 7) and control ( n = 6) according to the second principal component. ( I ) DiPRO1 upregulation in RMS and Ewing’s sarcoma (ES) cell lines. RNA-seq data were extracted from the DepMap portal (Broad Institute). The whisker plot shows median (Q2) ± Q3-Q1 ± 1.5x IQR of ZNF555 expression by cancer type or in all cancer types (ALL). *** ( P < 0.001) indicates a significant difference with ALL. ( J ) Stable knockdown of RMS cells was achieved by transduction of lentiviral vectors producing nontargeting shRNA (shCtl) or DiPRO1-targeting shRNAs (shDiPRO1). Cell cycle analysis of TE671/RMS cells lacking DiPRO1 was performed 48 and 72 h after transduction with vectors expressing DiPRO1-targeting shRNA. Transduced RMS cells were fixed in ethanol and stained with propidium iodide (PI). DNA content and percentage of cells in each cell cycle phase were determined by flow cytometry. The results represent two independent experiments. ( K ) RMS and myoblast (Myo) cells transfected with shCtl and shDiPRO1 were assayed for caspase-3 activity. The results were normalized to the protein quantity and appropriate shCtl was referenced as 100%. All conditions represent three or six transfections (RMS n = 6, Myo n = 3), proportions (%) ± SD. ( L ) DiPRO1 induces RMS cell proliferation. RMS cells were stably transduced with a retroviral vector expressing the DiPRO1 ORF (pDiPRO1) and compared with their parental counterparts (Ctl). The proliferation rate was estimated for DiPRO1-overexpressing RMS (pDiPRO1) and their control (Ctl) by Counting Kit 8 (Sigma-Aldrich). Cell number was determined using a titration curve performed at different cell dilutions from 0 to 25,000 cells/well. Cells were then seeded at 2.0 × 10 3 , 5.0 × 10 3 , 1.0 × 10 4 , and 1.5 × 10 4 cells/well and absorbance was measured at 460 nm after 72 h of proliferation. Viable cells were compared to the initial number of cells. Data were expressed as mean % relative to initial cell number ± SD and represent four independent experiments ( n = 4). The titration curve for RMS-Ctl is presented in Fig. . Data information: In ( A – C , E – H ), Global transcriptome analysis of microarray was implemented using total mRNA. The Limma R package was used for statistical analysis. In ( B , C , F , I ), boxplots represent median (line) ± dispersion, box length IQR = Q3-Q1, whisker length = 1.5 * IQR. In ( D , I – L ), Welch's two-sample t -test. In ( B – D , I , K , L ), *,**, and *** indicate significant differences with the corresponding control, P < 0.05, 0.01, and 0.001, respectively. .

Techniques: Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Expressing, Control, Staining, Flow Cytometry, shRNA, Knockdown, Functional Assay, Software, RNA Sequencing Assay, Whisker Assay, Cell Cycle Assay, Transfection, Activity Assay, Titration, Microarray, Dispersion

( A – I ) DiPRO1 knockdown was achieved by lentiviral transduction of vectors producing a non-targeted shRNA (shCtl) and a shRNA targeting the DiPRO1 gene (shDiPRO1). ( A ) The efficiency of DiPRO1 inhibition was verified by mRNA expression analysis in TE671 cells. Three different shRNAs (shDiPRO1-1/2/3) were tested individually and the inhibition effect was analyzed by RT-qPCR. Total mRNA was extracted 48 h after transduction. The results represent three independent assays and were compared to shCtl, referenced as 100%, using the t -test, * P < 0.05, n = 3, proportions (%) ± SD. ( B ) Induction of cell death in TE671 cells lacking DiPRO1 48 h after transduction. Three independent experiments correspond to three individual shDiPRO1. Transduced RMS cells were stained with propidium iodide (PI). The percentage of dead cells in non-gated areas was determined by flow cytometry. The pairwise t -test was applied for statistical analysis, * P < 0.05, n = 3, proportions (%) ± SD. ( C ) Dramatic cell death was observed 5–7 days after transduction of TE671 and JR RMS, and A673 and EW7 Ewing sarcoma cell lines. Scale bar: 50 µm. The cell images are also represented in Appendix Fig. . ( D ) Heatmap showing genes that were affected in RMS and Ewing sarcoma cells under DiPRO1 knockdown versus control. Clustering analysis of Euclidean distribution, full linkage. ( E ) Boxplots of DiPRO1 downregulation in both RMS and Ewing sarcoma cells with DiPRO1 knockdown (red) and in control (blue). Data were presented as median (Q2) ± Q3-Q1, whiskers extend to 1.5x IQR, ** P < 0.01. ( F ) Canonical pathways differentially regulated in cancer (RMS + ES, n = 9) and normal (Myo, n = 6–7) cells under DiPRO1 knockdown. Analysis was performed using Ingenuity software, Fisher’s Exact test p < 0.05. ( G ) Common apoptosis hallmarks in RMS and Ewing sarcoma (RMS + ES, n = 9), myoblasts (Myo, n = 6–7) and appropriate controls. ( H , I ) MCM6 protein ( H ) and gene ( I ) expression in myoblasts (Myo, n = 6–7) and cancer (RMS + ES, n = 9) cells with DiPRO1 KD and control counterparts was determined 48 h after transduction. The mRNA expression data are presented as median (Q2) ± Q3-Q1, whiskers extend to 1.5x IQR. * P < 0.05, *** P < 0.001. Western blot was performed using anti-MCM6 and anti-actin antibodies (internal control). Data information: In ( D – G , I ), global transcriptome analysis by microarray was implemented using total mRNA extracted from RMS and Ewing sarcoma cell lines: with DiPRO1 knockdown (shDiPRO1, n = 9) and corresponding controls (shCtl, n = 9). Differentially expressed genes versus controls ( P < 0.05) were analyzed using the Limma R package. .

Journal: EMBO Molecular Medicine

Article Title: DiPRO1 distinctly reprograms muscle and mesenchymal cancer cells

doi: 10.1038/s44321-024-00097-z

Figure Lengend Snippet: ( A – I ) DiPRO1 knockdown was achieved by lentiviral transduction of vectors producing a non-targeted shRNA (shCtl) and a shRNA targeting the DiPRO1 gene (shDiPRO1). ( A ) The efficiency of DiPRO1 inhibition was verified by mRNA expression analysis in TE671 cells. Three different shRNAs (shDiPRO1-1/2/3) were tested individually and the inhibition effect was analyzed by RT-qPCR. Total mRNA was extracted 48 h after transduction. The results represent three independent assays and were compared to shCtl, referenced as 100%, using the t -test, * P < 0.05, n = 3, proportions (%) ± SD. ( B ) Induction of cell death in TE671 cells lacking DiPRO1 48 h after transduction. Three independent experiments correspond to three individual shDiPRO1. Transduced RMS cells were stained with propidium iodide (PI). The percentage of dead cells in non-gated areas was determined by flow cytometry. The pairwise t -test was applied for statistical analysis, * P < 0.05, n = 3, proportions (%) ± SD. ( C ) Dramatic cell death was observed 5–7 days after transduction of TE671 and JR RMS, and A673 and EW7 Ewing sarcoma cell lines. Scale bar: 50 µm. The cell images are also represented in Appendix Fig. . ( D ) Heatmap showing genes that were affected in RMS and Ewing sarcoma cells under DiPRO1 knockdown versus control. Clustering analysis of Euclidean distribution, full linkage. ( E ) Boxplots of DiPRO1 downregulation in both RMS and Ewing sarcoma cells with DiPRO1 knockdown (red) and in control (blue). Data were presented as median (Q2) ± Q3-Q1, whiskers extend to 1.5x IQR, ** P < 0.01. ( F ) Canonical pathways differentially regulated in cancer (RMS + ES, n = 9) and normal (Myo, n = 6–7) cells under DiPRO1 knockdown. Analysis was performed using Ingenuity software, Fisher’s Exact test p < 0.05. ( G ) Common apoptosis hallmarks in RMS and Ewing sarcoma (RMS + ES, n = 9), myoblasts (Myo, n = 6–7) and appropriate controls. ( H , I ) MCM6 protein ( H ) and gene ( I ) expression in myoblasts (Myo, n = 6–7) and cancer (RMS + ES, n = 9) cells with DiPRO1 KD and control counterparts was determined 48 h after transduction. The mRNA expression data are presented as median (Q2) ± Q3-Q1, whiskers extend to 1.5x IQR. * P < 0.05, *** P < 0.001. Western blot was performed using anti-MCM6 and anti-actin antibodies (internal control). Data information: In ( D – G , I ), global transcriptome analysis by microarray was implemented using total mRNA extracted from RMS and Ewing sarcoma cell lines: with DiPRO1 knockdown (shDiPRO1, n = 9) and corresponding controls (shCtl, n = 9). Differentially expressed genes versus controls ( P < 0.05) were analyzed using the Limma R package. .

Techniques: Knockdown, Transduction, shRNA, Inhibition, Expressing, Quantitative RT-PCR, Staining, Flow Cytometry, Control, Software, Western Blot, Microarray

( A ) Heatmaps displaying size-sorted hypo- and hypermethylated DMC in shDiPRO1 versus shCtl. Overlapped myoblast (Myo) regions in RMS CGIs are colored blue and overlapped RMS regions in myoblast CGIs are colored red. The percentage of overlap is indicated. The bar reflects signal intensity. Y-axis: DNA fragments per 1 M reads per 1 K. X-axis: surrounding area corresponding to 500% of each CGI region, segmented into 200 bins. ( B ) DNA repeat class distribution within DiPRO1-linked hypomethylated DMC regions in RMS cells. ( C ) Hypomethylated DMC signals are overrepresented in chromosome 19. The analysis was performed using the positional gene enrichment (PGE) tool. ( D ) Hallmark enrichment of genes linked to differentially methylated genes (DMGs) was identified in myoblast and RMS cells following DiPRO1 knockdown. ( E – G ) Enrichment of TNFA Signaling via NFKB pathway in hypomethylated DMGs of RMS cells. ( E ) TNFSF9 and TRAF1 genes were hypomethylated in TSS regions and upregulated in RMS cells. The prediction analysis using the FIMO tool Version 5.5.4 identified the presence of potential DiPRO1 binding motifs within the GGI, aligning with the proximal enhancers. CGI, GC (%), cCRE (cis-regulatory elements), and DMC tracks over the TNFSF9 and TRAF1 loci. ( F ) Heatmap expression of the TNFA signaling via NFKB pathway gene set across control and DiPRO1 KD samples of RMS and Ewing sarcoma cells ( n = 9) and myoblasts ( n = 6–7). Clustering analysis of Euclidean distribution, complete linkage. ( G ) PCA analysis of DEGs of the TNFA signaling via NFKB pathway of DiPRO1 KD in RMS and Ewing sarcoma cells ( n = 9) and myoblasts ( n = 6–7). Data Information: In ( A – C , E ), methylation profiles were assessed using MIRA-seq in DNA samples from RMS TE671 and immortalized human myoblast (Myo) cells. The signals from methylation-enriched DNA (ENR) were normalized to unenriched input DNA (INP). CpG islands from cells expressing a DiPRO1-targeting shRNA (shDiPRO1) were compared with control cells expressing a nontargeting shRNA (shCtl), and differentially methylated CpG islands (DMC) were analyzed between normal and cancer muscle cells. In ( D , F , G ), global transcriptome analysis by microarray was implemented using total mRNA extracted from RMS and Ewing sarcoma cell lines: with DiPRO1 knockdown (shDiPRO1) and corresponding controls (shCtl). Three to six separate extractions from two independent experiments were performed to collect the replicates ( n = 9 per condition). Differentially expressed genes in shDiPRO1- cells versus corresponding controls ( P < 0.05) were analyzed using the Limma R package. CGI, CpG islands according to Gardiner-Garden and Frommer criteria. .

Journal: EMBO Molecular Medicine

Article Title: DiPRO1 distinctly reprograms muscle and mesenchymal cancer cells

doi: 10.1038/s44321-024-00097-z

Figure Lengend Snippet: ( A ) Heatmaps displaying size-sorted hypo- and hypermethylated DMC in shDiPRO1 versus shCtl. Overlapped myoblast (Myo) regions in RMS CGIs are colored blue and overlapped RMS regions in myoblast CGIs are colored red. The percentage of overlap is indicated. The bar reflects signal intensity. Y-axis: DNA fragments per 1 M reads per 1 K. X-axis: surrounding area corresponding to 500% of each CGI region, segmented into 200 bins. ( B ) DNA repeat class distribution within DiPRO1-linked hypomethylated DMC regions in RMS cells. ( C ) Hypomethylated DMC signals are overrepresented in chromosome 19. The analysis was performed using the positional gene enrichment (PGE) tool. ( D ) Hallmark enrichment of genes linked to differentially methylated genes (DMGs) was identified in myoblast and RMS cells following DiPRO1 knockdown. ( E – G ) Enrichment of TNFA Signaling via NFKB pathway in hypomethylated DMGs of RMS cells. ( E ) TNFSF9 and TRAF1 genes were hypomethylated in TSS regions and upregulated in RMS cells. The prediction analysis using the FIMO tool Version 5.5.4 identified the presence of potential DiPRO1 binding motifs within the GGI, aligning with the proximal enhancers. CGI, GC (%), cCRE (cis-regulatory elements), and DMC tracks over the TNFSF9 and TRAF1 loci. ( F ) Heatmap expression of the TNFA signaling via NFKB pathway gene set across control and DiPRO1 KD samples of RMS and Ewing sarcoma cells ( n = 9) and myoblasts ( n = 6–7). Clustering analysis of Euclidean distribution, complete linkage. ( G ) PCA analysis of DEGs of the TNFA signaling via NFKB pathway of DiPRO1 KD in RMS and Ewing sarcoma cells ( n = 9) and myoblasts ( n = 6–7). Data Information: In ( A – C , E ), methylation profiles were assessed using MIRA-seq in DNA samples from RMS TE671 and immortalized human myoblast (Myo) cells. The signals from methylation-enriched DNA (ENR) were normalized to unenriched input DNA (INP). CpG islands from cells expressing a DiPRO1-targeting shRNA (shDiPRO1) were compared with control cells expressing a nontargeting shRNA (shCtl), and differentially methylated CpG islands (DMC) were analyzed between normal and cancer muscle cells. In ( D , F , G ), global transcriptome analysis by microarray was implemented using total mRNA extracted from RMS and Ewing sarcoma cell lines: with DiPRO1 knockdown (shDiPRO1) and corresponding controls (shCtl). Three to six separate extractions from two independent experiments were performed to collect the replicates ( n = 9 per condition). Differentially expressed genes in shDiPRO1- cells versus corresponding controls ( P < 0.05) were analyzed using the Limma R package. CGI, CpG islands according to Gardiner-Garden and Frommer criteria. .

Techniques: Methylation, Knockdown, Binding Assay, Expressing, Control, shRNA, Microarray

( A ) Heatmaps (top) displaying SIX1 target signals from the GSEA data collection within CGIs of hg38, hypo-, and hypermethylated DMC in DiPRO1 KD RMS. The bar reflects the signal intensity. Y-axis: DNA fragments per 1 M reads per 1 K. X-axis: 10 Kb surrounding each region. Pie chart (bottom) displays overlapping regions. The ChIP-seq dataset of SIX1 KD in RMS cells were retrieved from GSE173155 and uploaded by the IGV browser. ( B , C ) The SIX1 KD RNA-seq (GSE173155) data of RMS cells were processed using DEBrowser, DESeq2 (TMM normalization, local fit, LRT test). Global transcriptome analysis by microarray was implemented using total mRNA extracted from myoblasts (Myo), RMS, and Ewing sarcoma (ES) cell lines with DiPRO1 knockdown (shDiPRO1) and corresponding controls (shCtl). Three to four separate extractions from two independent experiments were performed to collect the replicates. Differentially expressed genes in shDiPRO1- cells versus corresponding controls ( P < 0.05) were analyzed using the Limma R package. ( B ) Hallmark enrichment analysis of shared upregulated DEGs in SIX1 KD ( n = 4) and DiPRO1 KD in RMS (left, n = 3) and myoblasts (right, n = 6–7) using MSigDB database. ( C ) Jvenn diagram of the myogenesis gene set shows a joint signature of SIX1 KD and DiPRO1 KD, n = 3–7. ( D ) The skeletal muscle Aldolase A (ALDOA) gene containing an MEF3 motif in the promoter region was hypermethylated in DiPRO1 KD RMS cells at the DiPRO1 binding motif. CGI, DiPRO1 ChIP-seq, cCRE (candidate cis -regulatory elements) and hypermethylated DMC tracks over the ALDOA locus. ( E ) DiPRO1 binds directly to MEF3 motifs. Competition EMSA evaluation of the specific binding of DiPRO1 motif 1 (lanes 7–13) and MEF3 motifs from ALDOA (lanes 1–6) and MYOG (lane 14) with DiPRO1 protein from nuclear extract of RMS cells overexpressing pDiPRO1-HA. Binding specificity was examined by competition in the binding reaction between nuclear proteins and labeled oligoprobes by adding a 300-fold molar excess of homologous unlabeled probes (MEF3) or heterologous probes DiPRO1, MAF, 4qAe, TRAF1, and TNFSF9. In vitro-translated SIX1 protein was added to pDiPRO1-RMS nuclear extracts and incubated with DiPRO1 and ALDOA-MEF3 probes (lanes 6, 10). EMSA Supershift was performed by pre-incubating pDIPRO_HA RMS nuclear extract with anti-HA Ab. Band shifts 1 and 2 (BS 1/2) were specific for DiPRO1 and band shift 4 (BS4) was specific for SIX1. ( F ) The trans-acting properties of DiPRO1/SIX1/MEF3 complexes were investigated. Multimerized MEF3 motifs, positioned upstream of a minimal TATA box to drive a firefly luciferase reporter, were co-expressed with pDiPRO1 alone (200 ng) and/or with increasing amounts of the transcriptional activator pSix1VP16 (20 ng and 200 ng) in E18.5 Six1/4/5 KO primary mouse myoblasts. Luciferase expression was analyzed 48 h post-transfection. The data represent results from triplicates in two experiments ( n = 6), normalized against the renilla luciferase reporter vector, and the firefly luciferase expression with pTATA-box was set to 1. The data were shown as m±SD. Welch's two-sample t- test was applied for statistical difference. Boxplots are colored according to the average mean (indicated at the top). ( G , H ), scRNAseq datasets (GSE218974) derived from PDX primary RMS cultures ( n = 3 eRMS and n = 3 aRMS) were reanalysed using the Seurat R toolkit. The individual data were normalized, feature-selected, and integrated into the individual data samples using the SCTransform function. ( G ) Integrated UMAP plot showing SIX1 expression density across RMS cell populations. Analysis was performed using the R package scCustomize ( n = 6). ( H ) Coexpression curves for DiPRO1 and SIX1 genes in differentiated and cycling RMS cell populations. The stat_cor(method = “pearson”) R function was used. Pearson correlation coefficient (R) and p value represents the statistical significance of the linear relationship between two gene expression, n = 6. .

Journal: EMBO Molecular Medicine

Article Title: DiPRO1 distinctly reprograms muscle and mesenchymal cancer cells

doi: 10.1038/s44321-024-00097-z

Figure Lengend Snippet: ( A ) Heatmaps (top) displaying SIX1 target signals from the GSEA data collection within CGIs of hg38, hypo-, and hypermethylated DMC in DiPRO1 KD RMS. The bar reflects the signal intensity. Y-axis: DNA fragments per 1 M reads per 1 K. X-axis: 10 Kb surrounding each region. Pie chart (bottom) displays overlapping regions. The ChIP-seq dataset of SIX1 KD in RMS cells were retrieved from GSE173155 and uploaded by the IGV browser. ( B , C ) The SIX1 KD RNA-seq (GSE173155) data of RMS cells were processed using DEBrowser, DESeq2 (TMM normalization, local fit, LRT test). Global transcriptome analysis by microarray was implemented using total mRNA extracted from myoblasts (Myo), RMS, and Ewing sarcoma (ES) cell lines with DiPRO1 knockdown (shDiPRO1) and corresponding controls (shCtl). Three to four separate extractions from two independent experiments were performed to collect the replicates. Differentially expressed genes in shDiPRO1- cells versus corresponding controls ( P < 0.05) were analyzed using the Limma R package. ( B ) Hallmark enrichment analysis of shared upregulated DEGs in SIX1 KD ( n = 4) and DiPRO1 KD in RMS (left, n = 3) and myoblasts (right, n = 6–7) using MSigDB database. ( C ) Jvenn diagram of the myogenesis gene set shows a joint signature of SIX1 KD and DiPRO1 KD, n = 3–7. ( D ) The skeletal muscle Aldolase A (ALDOA) gene containing an MEF3 motif in the promoter region was hypermethylated in DiPRO1 KD RMS cells at the DiPRO1 binding motif. CGI, DiPRO1 ChIP-seq, cCRE (candidate cis -regulatory elements) and hypermethylated DMC tracks over the ALDOA locus. ( E ) DiPRO1 binds directly to MEF3 motifs. Competition EMSA evaluation of the specific binding of DiPRO1 motif 1 (lanes 7–13) and MEF3 motifs from ALDOA (lanes 1–6) and MYOG (lane 14) with DiPRO1 protein from nuclear extract of RMS cells overexpressing pDiPRO1-HA. Binding specificity was examined by competition in the binding reaction between nuclear proteins and labeled oligoprobes by adding a 300-fold molar excess of homologous unlabeled probes (MEF3) or heterologous probes DiPRO1, MAF, 4qAe, TRAF1, and TNFSF9. In vitro-translated SIX1 protein was added to pDiPRO1-RMS nuclear extracts and incubated with DiPRO1 and ALDOA-MEF3 probes (lanes 6, 10). EMSA Supershift was performed by pre-incubating pDIPRO_HA RMS nuclear extract with anti-HA Ab. Band shifts 1 and 2 (BS 1/2) were specific for DiPRO1 and band shift 4 (BS4) was specific for SIX1. ( F ) The trans-acting properties of DiPRO1/SIX1/MEF3 complexes were investigated. Multimerized MEF3 motifs, positioned upstream of a minimal TATA box to drive a firefly luciferase reporter, were co-expressed with pDiPRO1 alone (200 ng) and/or with increasing amounts of the transcriptional activator pSix1VP16 (20 ng and 200 ng) in E18.5 Six1/4/5 KO primary mouse myoblasts. Luciferase expression was analyzed 48 h post-transfection. The data represent results from triplicates in two experiments ( n = 6), normalized against the renilla luciferase reporter vector, and the firefly luciferase expression with pTATA-box was set to 1. The data were shown as m±SD. Welch's two-sample t- test was applied for statistical difference. Boxplots are colored according to the average mean (indicated at the top). ( G , H ), scRNAseq datasets (GSE218974) derived from PDX primary RMS cultures ( n = 3 eRMS and n = 3 aRMS) were reanalysed using the Seurat R toolkit. The individual data were normalized, feature-selected, and integrated into the individual data samples using the SCTransform function. ( G ) Integrated UMAP plot showing SIX1 expression density across RMS cell populations. Analysis was performed using the R package scCustomize ( n = 6). ( H ) Coexpression curves for DiPRO1 and SIX1 genes in differentiated and cycling RMS cell populations. The stat_cor(method = “pearson”) R function was used. Pearson correlation coefficient (R) and p value represents the statistical significance of the linear relationship between two gene expression, n = 6. .

Techniques: ChIP-sequencing, RNA Sequencing Assay, Microarray, Knockdown, Binding Assay, Labeling, In Vitro, Incubation, Electrophoretic Mobility Shift Assay, Luciferase, Expressing, Transfection, Plasmid Preparation, Derivative Assay

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