Journal: PLoS ONE

Article Title: Cellular MicroRNAs 498 and 320d Regulate Herpes Simplex Virus 1 Induction of Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication by Targeting RTA

doi: 10.1371/journal.pone.0055832

Figure Lengend Snippet: ( A ) . Luciferase reporter assay for screening miRNAs that target KSHV RTA 3′UTR. 293T cells were co-transfected with negative control nucleotide of miRNA ( Neg. Ctrl. ) or mimics of several miRNAs together with the pGL3-Luc-RTA 3′UTR luciferase reporter and assayed for luciferase activity. ** P <0.01 and *** P <0.001 for Student’s t-test versus Neg. Ctrl. group. ( B ) . Both miR-498 and miR-320d only inhibited the reporter activity of pGL3-RTA 3′UTR but not that of pGL3-Control construct. Luciferase activity was detected by co-transfection pGL3-Control or pGL3-RTA 3′UTR construct along with Neg. Ctrl., mimic of miR-498 ( miR-498 ) or miR-320d ( miR-320d ) for 24 h in 293T cells. The relative reporter activity levels of pGL3-RTA 3′UTR and pGL3-Control in the Neg. Ctrl. group were considered to be “1” for comparison, respectively. ** P <0.01 for Student’s t-test versus pGL3-RTA 3′UTR plus Neg. Ctrl. group . ( C ) . RT-qPCR analysis for validating the miRNA microarray data. MiR-498 and miR-320d expression in BCBL-1 cells infected with HSV-1 or Mock for 24 h was quantitated by RT-qPCR. Relative quantities of miRNAs expression were represented as 2 −ΔΔCt on the y axis. ** P <0.01 and *** P <0.001 for Student’s t-test versus Mock group. ( D ) . Inhibition of RTA protein expression by miR-498 and miR-320d. A genomic RTA expression vector pcDNA3.1−3×Flag-RTA-3′UTR bearing the full 3′UTR sequences was co-transfected with pEGFP and mimic of miR-498 or miR-320d into 293T cells for 48 h. Cells were collected and immunoblotted with the indicated antibodies. The relative level of RTA was determined by quantitative densitometry. Numbers labeled above the RTA band were the relative intensities of the bands compared to EGFP. The relative level of RTA in the Neg. Ctrl.+pcDNA3.1−3×Flag-RTA-3′UTR+pEGFP-N2 group was considered to be 1 for comparison. ( E ) . Schematic illustration of the putative seed sequences of miR-320d ( S1 ) and miR-498 ( S2 ) within the 3′UTR of RTA, and mutagenesis of target sites in the RTA 3′UTR or miRNA mimics. Mutated nucleotides in the target sites were framed in red. ( F ) . Effect of mutagenesis on miR-498 or miR-320d targeting of the 3′UTR of RTA. After co-transfection of RTA wild type ( RTA WT ) or mutant 3′UTR construct ( mut S1 or mut S2 ) together with natural (miR-498 or miR-320d) or mutant miR-498 or miR-320d mimic (mut miR-498 or mut miR-320d) for 24 h, 293T cells were assayed for luciferase activity. *** P <0.001 for Student’s t-test versus Neg.Ctrl.+RTA WT.

Article Snippet: Vero cells (African green monkey kidney fibroblasts) and 293T cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium (DMEM) +10% FBS.

Techniques: Luciferase, Reporter Assay, Transfection, Negative Control, Activity Assay, Control, Construct, Cotransfection, Comparison, Quantitative RT-PCR, Microarray, Expressing, Infection, Inhibition, Plasmid Preparation, Labeling, Mutagenesis

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–B) >4 weeks post-infection with Yptb ΔyopM, mice were injected i.v. with YopE69–77 peptide or control vehicle. At 18 hours post-injection, the total mAT was isolated for microarray analysis. (A) Pathway analysis was performed using Enrichr and graphed based on enrichment score (Log10 (adjusted p value)). (B) Diagonal plot with red dots representing genes from the 2 most significantly enriched lipid metabolic pathways (GO: 0046460 and GO: 0006368) from (A) that were downregulated ≥2 fold (with an adjusted p value <0.05) after peptide injection compared to control. (C–D) >4 weeks post-infection with the Yptb ΔyopM, mice were injected i.v. with YopE69–77 peptide or control vehicle and analyzed 4 hours post-injection. (C) Relative concentration of adiponectin in serum. (D) Relative concentration of cholesterol in serum. (E–F) Relative gene expression determined by RT-qPCR on adipocytes isolated from mAT of mice >4 weeks post infection with Yptb ΔyopM at 18 hours post-injection. (G–H) >4 weeks post-infection with Yptb ΔyopM, the mAT was cultured with YopE69–77 peptide or control vehicle and AT and culture supernatants were analyzed after 48 hours. (G) Relative gene expression was evaluated from the indicated genes. (H) Levels of free glycerol were measured in culture supernatants. Error bars in all graphs represent standard deviation. Data are representative of at least 5 experiments with 2–7 mice per group. ns not significant *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Figure S6.

Article Snippet: N/A Oligonucleotides 5’-ATGAGTAACTTCTCTGGATTTACG-3’ IDT YscF-Forward 5’-TTATGGGAACTTCTGTAGGATG-3’ IDT YscF-Reverse Acaca Qiagen QT00258419 Dgat2 Qiagen QT00134477 Fasn Qiagen QT00149240 Gbp4 Qiagen QT00174608 Gbp11 Qiagen QT01255142 Ifi47 Qiagen QT00116935 Pparg Qiagen QT00100296 Hprt Qiagen QT00166768 Recombinant DNA Software and Algorithms clusterProfiler Yu et al., 2012 N/A Cytoscape 3.4.0 Nepusz et al., 2012 N/A DEseq pipeline Anders and Huber, 2010 N/A Enrichr http://amp.pharm.mssm.edu/Enrichr/ N/A FastQC software package version 0.11.5 Babraham Bioinformatics N/A Flowjo software Treestar N/A Imaris software Bitplane N/A Prism software Graphpad N/A STAR aligner Dobin et al., 2013 N/A Trimmomatic Bolger et al., 2014 N/A Other Open in a separate window DATA AND SOFTWARE AVAILABILITY

Techniques: Infection, Injection, Control, Isolation, Microarray, Concentration Assay, Gene Expression, Quantitative RT-PCR, Cell Culture, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: DATA AND SOFTWARE AVAILABILITY

Article Snippet: N/A Oligonucleotides 5’-ATGAGTAACTTCTCTGGATTTACG-3’ IDT YscF-Forward 5’-TTATGGGAACTTCTGTAGGATG-3’ IDT YscF-Reverse Acaca Qiagen QT00258419 Dgat2 Qiagen QT00134477 Fasn Qiagen QT00149240 Gbp4 Qiagen QT00174608 Gbp11 Qiagen QT01255142 Ifi47 Qiagen QT00116935 Pparg Qiagen QT00100296 Hprt Qiagen QT00166768 Recombinant DNA Software and Algorithms clusterProfiler Yu et al., 2012 N/A Cytoscape 3.4.0 Nepusz et al., 2012 N/A DEseq pipeline Anders and Huber, 2010 N/A Enrichr http://amp.pharm.mssm.edu/Enrichr/ N/A FastQC software package version 0.11.5 Babraham Bioinformatics N/A Flowjo software Treestar N/A Imaris software Bitplane N/A Prism software Graphpad N/A STAR aligner Dobin et al., 2013 N/A Trimmomatic Bolger et al., 2014 N/A Other Open in a separate window DATA AND SOFTWARE AVAILABILITY

Techniques: Software, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, SYBR Green Assay, Staining, Microarray

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: GenomeGraph of SKAR as a splice target of TDP-43. HEK293E cells were transfected with control siRNA (scrambled) or treated with siRNA against TDP-43 (siRNA TDP-43 ). Four biological replicates of each group were hybridized on a Human Exon 1.0-ST Gene Chip. Intensity values of microarray hybridizations, single values (gray), mean group intensities of scrambled siRNA (blue) and siRNA TDP-43 (green), are shown as normalized background-corrected logarithmic intensities ( A ) and RMA corrected probe-level data ( B ). Vertical lines separate the 18 individual probe sets covering the POLDIP3/SKAR gene. ( C ) Depicted are the mean group values of the FIRMA score. The fold change of the FIRMA score (FC(F)) is shown in red. ( D ) Genomic representation of the POLDIP3/SKAR gene in orange. Gray lines at the top of this panel indicate localization of the individual probe sets within the genomic coordinates. ( E ) The two Ensembl annotated alternative splice isoforms SKAR α and SKAR β are depicted in blue. SKAR exon 3 is highlighted by a box. ( F ) The SKAR α protein isoform is shown in pink, the RRM domain is shown in dark blue. Highlighted in green is the exon 3 derived part. At the bottom the amino acid sequence of exon 3 is given.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Microarray, Derivative Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: Validation of SKAR alternative splicing upon transient silencing of TDP-43. TDP-43 was either silenced transiently by siRNA treatment ( A , C , E and G ) or stably by use of lentiviral particles encoding for a TDP-43-specific shRNA followed by the selection of single cell clones ( B , D and F ). For transient silencing, HEK293E cells were either mock treated (m) or transiently transfected with scrambled control siRNA (scr), with one of four different TDP-43-specific siRNAs (siRNA TDP-43 A-D) or with one of five specific siRNAs against FUS (siRNA FUS A-E), as indicated. (A–D) Total RNA was extracted and analyzed by RT–PCR. (A and B) Semi-quantitative RT–PCR was performed with primer pairs specific for TDP-43, SKAR (ex2–ex4), SKAR α (ex2|3–ex4) and SKAR β (ex2|4–ex4). (C and D) Real-time PCR was performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKARα/PBGD, SKARβ/PBGD and total SKAR/PBGD ratios were recalculated into absolute copy values and normalized to total SKAR values. Shown are the mean values of five independent experiments ± SEM. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. Original qRT–PCR data is presented in Supplementary Figure S1A and S1B , respectively. (E–G) Protein was extracted, electrophoresed and resulting western blots probed with antibodies specific for TDP-43, SKAR (both isoforms) and SKAR α. GAPDH was used as a loading control. FUS silencing efficiency was controlled by use of an anti-FUS antibody. Note, that, depending on the primer pair and antibody used, SKAR RNA and protein isoforms, respectively, are visualized as two bands with different molecular weights. The upper band represents SKAR α, the lower corresponds to SKAR β, as indicated.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Selection, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR alternative splicing is dependent on RRM1 of TDP-43. ( A ) Stably silenced HEK293E cells (shRNA TDP-43 ) or transiently silenced HEK293 cells (siRNA TDP-43 ) were transiently transfected with either control vector (−) or Flag-TDP-43 variants (wt, ΔRRM1, ΔRRM2, ΔRRM1/2, FFLL and ΔGRD or disease-associated mutations, as indicated). Parental HEK293E cells or cells treated with a scrambled siRNA (−) were used as an internal control. (A) Total RNA was extracted and subjected to semi-quantitative RT–PCR using primer pairs amplifying total TDP-43, endogenous TDP-43, total SKAR (ex2–ex4), SKAR α (ex2|3–ex4), SKAR β (ex2|4–ex4) and PBGD as a housekeeping gene. ( B and E ) RNA was extracted and real-time PCR performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKAR α/PBGD, SKAR β/PBGD and total SKAR/PBGD ratios were re-calculated into absolute copy values and normalized to total SKAR values. Original qRT data is presented in Supplementary Figure S1C and S1D , respectively. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. ( C and D ) Protein was extracted, electrophoresed and resulting western blots probed with anti-TDP-43, anti-Flag and anti-SKAR antibodies. GAPDH was used as a loading control. (D) Shown are the mean values ± SEM of densitometric analysis of three independent experiments. * P < 0.05; ** P < 0.005; ns = not significant.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: A repeat containing RNA stretch 3′ of exon 3 is necessary for TDP-43 and SKAR splicing. ( A ) Schematic representation of constructs used for refined RNA crosslinking assays. ( B ) Indicated fragments of SKAR DNA were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt or FFLL. No RNA was added to control samples. Samples were UV crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates (left panel) were probed with anti-TDP-43 and anti-Flag to visualize co-precipitated endogenous and exogenous TDP-43. Biotinylated SKAR RNAs pulled down transfected as well as endogenous TDP-43 wt but not FFLL. Protein inputs (right panel) of HEK293E lysates confirmed even transfection efficiencies. ( C ) Schematic representation of the three repeat motifs and mutagenized variants within the SKAR pre-RNA 3′ of exon 3. ( D ) Non-mutated or mutagenized variants of SKAR DNA part-5 were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt. No RNA was added to control samples. Samples were UV-crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates were probed with anti-TDP-43 and anti-Flag to visualize coprecipitated endogenous and exogenous TDP-43. ( E ) Schematic representation of the used SKAR minigene construct pTB SKAR part-3/4/5. Primer annealing sites are indicated by arrows. ( F and G ) HEK293E cells were transfected with pTB SKAR part-3/4/5 variants, as indicated. RNA was extracted and used for RT–PCR using primers for pTB and PBGD as a housekeeping gene. (F) Representative RT–PCR is shown. (G) Shown are the results (mean values ± SEM) of densitometric analysis of seven independent experiments calculated as the ratio of SKAR α to SKAR β. * P < 0.05; *** P < 0.0005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Construct, In Vitro, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR β is more active than SKAR α and leads to enhanced translation and increased cell size. ( A ) HEK293E cells were treated with control siRNA or transfected with siRNA against SKAR or TDP-43 as indicated. Stably silenced siRNA TDP-43 and transiently transfected HEK293E cells were transfected with either control vector (−) or plasmids encoding for Myc-SKAR α, Myc-SKAR β or Flag-TDP-43 wt, as indicated. Cells were serum starved for 16 h. After 6 h serum-stimulation cells were harvested, protein extracted and electrophoresed. Resulting western blots were probed with anti-SKAR, anti-phospho S6K1 (Thr389), anti-S6K1, anti-phospho S6 (Ser235/236), anti-S6, anti-phospho Akt substrate (RXRXXS/T) and anti-TDP-43 antibodies. GAPDH was used as a loading control. Transfection of SKAR β or depletion of TDP-43 results in overall stronger phospho-signal compared to SKAR α. ( B ) Schematic representation of luciferase constructs used for analysis of translation. ( C–G ) HEK293E cells were transfected with either Myc-SKAR α or Myc-SKAR β (C) or with control siRNA (scr) and individual siRNA TDP-43 A–D, as indicated (D–G). (C–E) Before DNA/siRNA transfection, cells were transfected with firefly control vector plus either intron-containing or intron-less Renilla luciferase constructs. (C and D) Luciferase activity was measured and normalized to control treated HEK293E cells. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. Western blotting confirmed equal expression of Myc-SKAR α and Myc-SKAR β (C, right panel). (E) qRT–PCR confirmed equal RNA levels of Renilla and firefly luciferase in non-silenced and silenced HEK293E cells. (F) Cells were counted and equal numbers of cells was collected. Protein amount was determined using BCA protein assay. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. (G) Cell size was analyzed by flow cytometry, monitoring the forward scatter parameter. Shown are the mean values ± SEM of five independent experiments. * P < 0.05; ** P < 0.005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Stable Transfection, Plasmid Preparation, Western Blot, Luciferase, Construct, Activity Assay, Expressing, Quantitative RT-PCR, Bicinchoninic Acid Protein Assay, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Melanin synthesis causes γH2AX foci formation DNA strand breaks and abasic sites formation. ( A ) Immunofluorescence of B16 cells treated with PTU and tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI (blue) and γH2AX in (red). Experiment was performed with two biological replicates and a representative image is depicted. Scale bar 10 μm. ( B ) Quantitation of mean fluorescence intensity per cell of γH2AX from two biological replicates of pigmented day 7 PTU or tyrosine treated cells (shown in A). Data represented as a box plot, horizontal line represents mean and whiskers represent SEM. Ordinary one-way ANOVA was performed for multiple comparisons. Adjusted P -value: * P -value < 0.05, *** P -value < 0.001, **** P -value < 0.0001. ( C ) (Top) Cell pellet of day 7 B16 mouse melanoma cells grown at low density (100 cells/cm 2 ). Cells were left untreated for control treated with tyrosinase inhibitor 200 μM phenylthiourea (PTU) or 1mM tyrosinase substrate L-tyrosine (Tyr) for 7 days. Number of cells, mean ± SEM across three biological replicates is depicted below the image of the cell pellet. Numbers represent mean ± SEM cell counts across biological triplicates. (Bottom) Number of abasic sites in the genomic DNA was estimated by an aldehyde specific conjugation of biotin and subsequent detection using streptavidin based detection. Using standards, abasic sites per 10 5 bp is estimated. Bars represent mean ± SEM across duplicate biological experiments, each conducted in triplicates. Ordinary one-way ANOVA was performed for multiple comparisons * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001. ( D ) Single cell electrophoresis followed by comet analysis of B16 cells undergoing varying levels of pigmentation in the presence of PTU and tyrosine (alkaline comet assay). Experiment was carried out at mid phase (day 5) and late phase (day 7) of pigmentation. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed. Adjusted P values; ns non-significant, * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001, **** P -value < 0.0001. ( E ) Neutral comet assay on B16 unpigmented (day 0) and pigmented (day 7) cells. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Student's unpaired t-test was performed. P values ns non-significant. ( F ) Single cell electrophoresis followed by comet analysis of B16 cells untreated, treated with DMSO for 24 h, melanin synthesis ( ex-cellulo L-tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM dihydroxyindole (DHI) for 24 h (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05, **** P -value < 0.00001.

Article Snippet: Polκ (ab57070), γH2AX (CST 9718), total H2AX (CST 2595), pChk1 (CST 2348P), Total Chk1 (Invitrogen PA512096), pATR (CST 2853), total ATR (CST 2790), p53 (ab38497), p21 (CST 9706), PCNA (SC56), DCT (ab74073), Tyrosinase (custom synthesized genscript), mouse anti-BrdU antibody (ab136650) and Rat anti-BrdU antibody (ab6326).

Techniques: Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Conjugation Assay, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Neutral Comet Assay

Journal: Nucleic Acids Research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Normal human epidermal melanocytes (NHEM) respond to pigmentation induced DNA breaks by elevating Polκ. ( A ) NHEM cells were treated with 200 μM PTU or 1mM tyrosine for 7 days for differential pigmentation. (Top) Cell pellet, (bottom) western blot analysis of cell lysates with POLK, HSC70, phosphorylated H2AX, total H2AX and beta actin antibodies. Numbers below the blot correspond to control normalized expression of the indicated protein. Experiments were performed in biological duplicates. ( B ) Immunofluorescence of NHEM treated with PTU or tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI (blue) and γH2AX in (red). Experiments were performed with two biological replicates. Scale bar 10 μm. ( C ) Quantitation of mean fluorescence intensity per cell of γH2AX from two biological replicates of NHEM treated with PTU or tyrosine (shown in B). Ordinary one-way ANOVA was performed for multiple comparisons. Adjusted P values: * P -value < 0.05, **** P -value < 0.0001. ( D ) PTU and tyrosine treated NHEM cells were subjected to single cell electrophoresis and comet analysis (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: ** P -value < 0.001, **** P -value < 0.00001. ( E ) Heat map of expression (fold change) in mRNA levels of top two translesion polymerases (that were enriched in B16 microarray) (top), and a panel of known DNA replication stress response genes by qRT-PCR analysis in NHEM (Control, PTU or tyrosine treated). Data represented as mean of triplicate biological experiments. ( F ) Western blot analysis of NHEM treated with DMSO or 50 nM AZ20, a selective inhibitor of ATR kinase, for 24 h. Numbers below the blot correspond to control normalized expression of the indicated protein wrt beta-actin. Experiments were performed in biological duplicates. ( G ) mRNA levels of Polk in unpigmented B16 cells mock transfected, or with either control DNA, melanin modified DNA (plasmid DNA was incubated with L-DOPA and tyrosinase and column purified after 24 h) (Mel + DNA), DNA mixed with pre-synthesized melanin and coulmn purified [DNA+(Mel)], in-vitro melanin synthesis ( ex-cellulo l -tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM DHI (DHI) for 24 h. Bars represent percent mRNA levels compared to control across biological triplicates. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05. (Inset) Western blot analysis of B16 cells transfected with only DNA (Con DNA) or melanin-modified DNA (Mel + DNA) with Polκ antibody normalized to HSC70. Experiments were performed in biological triplicates.

Article Snippet: Polκ (ab57070), γH2AX (CST 9718), total H2AX (CST 2595), pChk1 (CST 2348P), Total Chk1 (Invitrogen PA512096), pATR (CST 2853), total ATR (CST 2790), p53 (ab38497), p21 (CST 9706), PCNA (SC56), DCT (ab74073), Tyrosinase (custom synthesized genscript), mouse anti-BrdU antibody (ab136650) and Rat anti-BrdU antibody (ab6326).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Microarray, Quantitative RT-PCR, Transfection, Modification, Plasmid Preparation, Incubation, Purification, Synthesized, In Vitro

Journal: Nucleic Acids Research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Silencing of Polκ during pigmentation prevents replication stress response despite elevated DNA damage. ( A ) Cell pellets of control non-targeting (shNT) and Polκ silenced (shPolκ) B16 cells on day 0 (left) and day 7 (right) of pigmentation. ( B ) Immunofluorescence analysis of day 7 pigmented shNT and shPolκ cells with phosphorylated H2AX antibody (puncta labelled in green) and the nucleus is counterstained with DAPI (blue). Scale bars represent 10 μm. ( C ) Quantitation of mean fluorescence intensity of γH2AX (shown in B) from two biological replicates of shNT and shPolκ cells across day 0, 5 and 7 of pigmentation induction. Two-way ANOVA was performed for multiple comparisons. Adjusted P values * P -value < 0.05 *** P -value < 0.0005 **** P -value < 0.00001 ns non-significant. ( D ) shNT and shPolκ expressing pigmented B16 cells were subjected to single cell electrophoresis and comet analysis (alkaline comet) on days 0, 5 and 7 of pigmentation induction. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed for multiple comparisons. Adjusted P values, ns non-significant, * P -value < 0.05. ( E ) Growth curve analysis of shNT and shPolκ expressing B16 cells on days 0, 5, 6 and 7 of pigmentation. Each point represents mean ± SEM across biological triplicates. Two-way ANOVA was performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( F ) Western blot analysis of DNA repair and cell cycle related proteins in shNT and shPolκ cells. Numbers below represent tubulin normalized fold changes wrt shNT. Experiments were performed in biological duplicates. ( G ) shNT and shPolκ expressing B16 cells were injected inside the flank of C57/BL6 mice and allowed to grow as tumors. The volume of the tumor was non-invasively monitored and plotted over time of biological triplicates mean ± SEM. Two-way ANOVA was performed for multiple comparisons. Adjusted P values: * P -value < 0.05. ( H ) Heat map of expression (fold change) in mRNA levels of a panel of known DNA replication stress response genes by qRT-PCR analysis in shNT (day 0-unpigmented and day 7-pigmented) and shPolκ (day 0-unpigmented and day 7-pigmented) B16 cells. ( I ) Western blot images and analysis of p-RPA2 and total RPA2 in shNT and shPolκ B16 cells (day 0 unpigmented and day 7 pigmented). Numbers below represent beta-actin normalized fold changes wrt shNT at day 0. Experiments were performed in biological triplicates. ( J ) Analysis of melanoma samples from TCGA data for mRNA expression of POLK (high, low or not detected) segregated into bar plots and proportion of mutations were plotted on y-axis. ( K ) Survival plot of melanoma patients with low or high expression of POLK from TCGA data. Analysis from Human Protein Atlas database. Paired t -test P value 0.017.

Article Snippet: Polκ (ab57070), γH2AX (CST 9718), total H2AX (CST 2595), pChk1 (CST 2348P), Total Chk1 (Invitrogen PA512096), pATR (CST 2853), total ATR (CST 2790), p53 (ab38497), p21 (CST 9706), PCNA (SC56), DCT (ab74073), Tyrosinase (custom synthesized genscript), mouse anti-BrdU antibody (ab136650) and Rat anti-BrdU antibody (ab6326).

Techniques: Immunofluorescence, Quantitation Assay, Fluorescence, Expressing, Electrophoresis, Western Blot, Injection, Quantitative RT-PCR

Journal: medRxiv

Article Title: Inhibition of SHP2 ameliorates psoriasis by decreasing TLR7 endosome localization

doi: 10.1101/2020.09.28.20202861

Figure Lengend Snippet: (A) Expression of PTPN11 ( gene encoding SHP2 ) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905). (B) Expression of PTPN11 in human PBMCs from psoriatic patients (n=14) and normal controls (n=16). (C) Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls. (D) Representative SHP2 staining in skin sections from psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm. (E) The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n=25) and normal controls (n=25). (F) Representative p-ERK staining of skin sections from psoriatic patients and normal controls. Scale bars: 200 μm. (G) Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back from C57BL/6J mice at day 5 (n=6/group). Data were normalized to GAPDH expression. (H) Representative histological sections of IMQ-treated or non-treated dorsal back from C57BL/6J mice at day 5. Scale bar: 100 μm. Data represent mean ± SEM. P values are determined by Two-tailed Mann-Whitney U test (A and B) or Two-tailed Student’s t test (E and G). * P <0.05, ** P <0.01.

Article Snippet: For immunohistochemistry, the human and mouse skin paraffin sections were deparaffinized, rehydrated, and antibody retrieved with sodium citrate, blocked, then stained with anti-SHP2 (Santa Cruz, catalog sc-7384), anti-ERK (Cell Signal Technology, catalog 4695), anti-CD68 (Cell Signal Technology, catalog 76437), anti-p-p65 (Cell Signal Technology, catalog 3033), anti-Ki67 (Abcam, catalog ab15580) were used at 1:100 overnight at 4°C.

Techniques: Expressing, Microarray, Western Blot, Derivative Assay, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Immunity

Article Title: CRISPR screens unveil nutrient-dependent lysosomal and mitochondrial nodes impacting intestinal tissue-resident memory CD8 + T cell formation

doi: 10.1016/j.immuni.2024.09.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified naive OT-I, P14, or YopE-I cells were activated for 20 h with 5 μg/ml plate-bound anti-CD3 (2C11, Bio X Cell) and 5 μg/ml plate-bound anti-CD28 (37.51, Bio X Cell) in complete Click’s medium (catalog #9195, Irvine Scientific) containing 10% fetal bovine serum (FBS; R&D Systems), 1× penicillin–streptomycin–L-glutamine (catalog #15140122, Thermo Fisher Scientific), and 55 μM β-mercaptoethanol.

Techniques: Purification, Virus, Expressing, Mutagenesis, Recombinant, Electron Microscopy, Control, Modification, Plasmid Preparation, Cell Isolation, Transfection, Sample Prep, Reverse Transcription, SYBR Green Assay, Microarray, RNA Sequencing Assay, Knock-In, Sequencing, Software, Flow Cytometry, Microscopy, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b -dependent mechanism

doi: 10.1152/ajpcell.00335.2019

Figure Lengend Snippet: Interferon-γ (Ifnγ) suppresses sirtuin 6 (Sirt6) expression. Young adult mouse colonocyte cells were treated with murine Tnfα (100 ng/mL), Ifnγ (100 ng/mL), Il6 (50 ng/mL), Il22 (50 ng/mL), Il1β (10 ng/mL), or culture medium (control) for 24 h. Total cell lysates were prepared. Sirt6 protein expression was measured by immunoblotting. Top: autoradiograph of a representative immunoblot. Bottom: densitometric analysis of the immunoblot data (normalized to β-actin). The experiments were performed three times. Results are expressed as means ± SE; n = 3 in each group. ****P < 0.0001 vs. control. The digital image of the immunoblots was adjusted with Photoshop software. The adjustments to the digital images do not alter the information contained therein. [For Source Data, see endnote.]

Article Snippet: Rabbit polyclonal antibody against mouse Sirt6 isoform 1 (NCBI RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_181586","term_id":"1821619512","term_text":"NM_181586"}} NM_181586 .3/ {"type":"entrez-protein","attrs":{"text":"NP_853617","term_id":"31712018","term_text":"NP_853617"}} NP_853617 .1, 1:1,000, catalog no. 12486; Cell Signaling Technology, Danvers, MA) and HRP-conjugated mouse monoclonal antibody against β-actin (1:50,000, catalog no. A3854; Sigma-Aldrich, St. Louis, MO) were used for the assay. miRNA microarray analysis.

Techniques: Expressing, Western Blot, Autoradiography, Software

Journal: American Journal of Physiology - Cell Physiology

Article Title: Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b -dependent mechanism

doi: 10.1152/ajpcell.00335.2019

Figure Lengend Snippet: Interferon-γ (Ifnγ) attenuates expression of sirtuin 6 (Sirt6) protein but not mRNA in mouse intestinal epithelial cells. A and B: dose-response experiment to characterize the effect of Ifnγ on Sirt6 gene expression in young adult mouse colonocyte (YAMC) cells. YAMC cells were treated with various concentrations of Ifnγ for 24 h. Sirt6 expression was measured by RT-qPCR (A) and immunoblotting (B). An autoradiograph of a representative immunoblot is shown in B. The results of three independent experiments were plotted after normalizing to β-actin; n = 3 in each group. *P < 0.05 versus 0. C and D: time-course experiment performed by continuous incubation of YAMC cells for different time points in the presence of 100 ng/mL Ifnγ. Sirt6 expression was measured by RT-qPCR (C) and immunoblotting (D). An autoradiograph of a representative immunoblot is shown in D. After normalizing to β-actin, the results of three independent experiments were plotted. Values are means ± SE; n = 3 in each group. *P < 0.05 versus 0. The digital images were adjusted with Photoshop software. The adjustments to the digital images do not alter the information contained therein. [For Source Data, see endnote.]

Article Snippet: Rabbit polyclonal antibody against mouse Sirt6 isoform 1 (NCBI RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_181586","term_id":"1821619512","term_text":"NM_181586"}} NM_181586 .3/ {"type":"entrez-protein","attrs":{"text":"NP_853617","term_id":"31712018","term_text":"NP_853617"}} NP_853617 .1, 1:1,000, catalog no. 12486; Cell Signaling Technology, Danvers, MA) and HRP-conjugated mouse monoclonal antibody against β-actin (1:50,000, catalog no. A3854; Sigma-Aldrich, St. Louis, MO) were used for the assay. miRNA microarray analysis.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Autoradiography, Incubation, Software

Journal: American Journal of Physiology - Cell Physiology

Article Title: Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b -dependent mechanism

doi: 10.1152/ajpcell.00335.2019

Figure Lengend Snippet: Interferon-γ (Ifnγ) induces expression of miR-92b, which targets the sirtuin 6 (Sirt6) 3′-untranslated region (UTR). A: profiling the effect of Ifnγ on microRNA (miRNA) expression in intestinal epithelial cells by miRNA microarray analysis. The Affymetrix GeneChip miRNA Array v. 4.0 was performed in triplicate on young adult mouse colonocyte (YAMC) cells at 12 h after indicated treatment. The heat map depicts miRNAs with a statistically significant change in expression following Ifnγ (100 ng/mL) treatment in YAMC cells. Control cells were treated with culture medium alone. A P value <0.05 and a 1.25-fold change cut off was applied. Color was assigned to each miRNA based on the change (in log2-fold change annotation) of relative expression across samples at the indicated treatments. B: predicted miR-92b target site in the mouse Sirt6 3′-UTR . C: qRT-PCR validation of the effect of Ifnγ on miR-92b expression in intestinal epithelial cells. YAMC cells were treated with Ifnγ for indicated times followed by measurement of miR-92b expression using the TaqMan advanced miRNA expression assay. Expression of miR-92b was normalized to the expression of miR-26a-5p (a recommended internal control miRNA). Values are means ± SE and represent average of findings from three independent experiments in each group. *P < 0.05 and **P < 0.01 versus control.

Article Snippet: Rabbit polyclonal antibody against mouse Sirt6 isoform 1 (NCBI RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_181586","term_id":"1821619512","term_text":"NM_181586"}} NM_181586 .3/ {"type":"entrez-protein","attrs":{"text":"NP_853617","term_id":"31712018","term_text":"NP_853617"}} NP_853617 .1, 1:1,000, catalog no. 12486; Cell Signaling Technology, Danvers, MA) and HRP-conjugated mouse monoclonal antibody against β-actin (1:50,000, catalog no. A3854; Sigma-Aldrich, St. Louis, MO) were used for the assay. miRNA microarray analysis.

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal: American Journal of Physiology - Cell Physiology

Article Title: Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b -dependent mechanism

doi: 10.1152/ajpcell.00335.2019

Figure Lengend Snippet: miR-92b directly targets sirtuin 6 (Sirt6) 3′-untranslated region (UTR). A: schematic diagram of pmirGLOmSirt6–3′-UTR, a dual-luciferase reporter construct containing the murine Sirt6 3′-UTR sequence. B: Sirt6 3′-UTR is a functional unit for maintaining homeostasis of Sirt6 mRNA expression. Young adult mouse colonocyte (YAMC) cells were transfected with indicated pmirGLO-based luciferase reporter constructs. After transfection (24 h), the cells were lysed followed by a dual-luciferase assay. C: interferon-γ (Ifnγ) inhibits pmirGLOmSirt6–3′-UTR activity. YAMC cells were transfected with pmirGLOmSirt6–3′-UTR and pmirGLO. After 24 h, the transfected cells were treated with Ifnγ or culture medium for an additional 24 h. Cells were processed for dual-luciferase assay as described in materials and methods. D: miR-92b mimic inhibits Sirt6 3′-UTR activity through recognizing its binding sequence. YAMC cells were transiently cotransfected with indicated luciferase reporter constructs and miR-92b mimic. As a control, cells were cotransfected with indicated luciferase reporter constructs and scrambled mimic. After 24 h, cells were lysed followed by a dual-luciferase assay. For luciferase analyses, values are means ± SE and represent average of findings from three independent experiments in each group. *P < 0.05, **P < 0.01, and ***P < 0.001. pGLO-3′-UTRWT indicates the wild-type pmirGLOmSirt6–3′-UTR luciferase reporter construct, whereas pGLO-3′-UTRMutated indicates the mutated pmirGLOmSirt6–3′UTR luciferase reporter construct.

Article Snippet: Rabbit polyclonal antibody against mouse Sirt6 isoform 1 (NCBI RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_181586","term_id":"1821619512","term_text":"NM_181586"}} NM_181586 .3/ {"type":"entrez-protein","attrs":{"text":"NP_853617","term_id":"31712018","term_text":"NP_853617"}} NP_853617 .1, 1:1,000, catalog no. 12486; Cell Signaling Technology, Danvers, MA) and HRP-conjugated mouse monoclonal antibody against β-actin (1:50,000, catalog no. A3854; Sigma-Aldrich, St. Louis, MO) were used for the assay. miRNA microarray analysis.

Techniques: Luciferase, Construct, Sequencing, Functional Assay, Expressing, Transfection, Activity Assay, Binding Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b -dependent mechanism

doi: 10.1152/ajpcell.00335.2019

Figure Lengend Snippet: miR-92b mimic suppresses sirtuin 6 (Sirt6) expression and miR-92b inhibitor rescues Sirt6 expression in interferon-γ (Ifnγ)-treated young adult mouse colonocyte (YAMC) cells. A: effect of an miR-92b mimic on Sirt6 expression in intestinal epithelial cells. YAMC cells were transfected with miR-92b mimic or scrambled mimic as indicated. After transfection (48 h), cells were lysed followed by immunoblotting to measure Sirt6 protein expression. B: inhibition of miR-92b attenuates the effect of Ifnγ on Sirt6 expression in intestinal epithelial cells. YAMC cells were transfected with miR-92b inhibitor or scrambled inhibitor as indicated. After transfection (24 h), cells were treated with Ifnγ (100 ng/mL) for another 24 h. At the end of treatment, cells were lysed and processed for immunoblotting to measure Sirt6 expression. Top: autoradiograph of a representative immunoblot. Bottom: densitometric analysis of the immunoblot data (normalized to β-actin). The experiments were performed three times. Results are expressed as means ± SE; n = 3 in each group. *P < 0.05. The digital image was adjusted with Photoshop software. The adjustments to the digital images do not alter the information contained therein. [For Source Data, see endnote.]

Article Snippet: Rabbit polyclonal antibody against mouse Sirt6 isoform 1 (NCBI RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_181586","term_id":"1821619512","term_text":"NM_181586"}} NM_181586 .3/ {"type":"entrez-protein","attrs":{"text":"NP_853617","term_id":"31712018","term_text":"NP_853617"}} NP_853617 .1, 1:1,000, catalog no. 12486; Cell Signaling Technology, Danvers, MA) and HRP-conjugated mouse monoclonal antibody against β-actin (1:50,000, catalog no. A3854; Sigma-Aldrich, St. Louis, MO) were used for the assay. miRNA microarray analysis.

Techniques: Expressing, Transfection, Western Blot, Inhibition, Autoradiography, Software

Journal: Carcinogenesis

Article Title: TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3

doi: 10.1093/carcin/bgw093

Figure Lengend Snippet: TGF-β-ALK5-Smad signaling induces CYR61 expression in pancreatic stellate cells. (A–C) Linear regression was performed using the microarray dataset GDS4103. n = 39 patient samples: (A) TGFB1, (B) SERPINE1 and (C) SMAD7. (D) Western blot of CYR61 (Abcam) in LTC-14 and imPSC cells that were serum starved in 1% FBS then treated with indicated doses of TGF-β1 for 16h. (E) Western blot of CYR61 (Abcam) in LTC-14 cells that were serum starved in 1% FBS then treated with 100 pM TGF-β for indicated times. (F) Quantitative RT-PCR for rCYR61 performed on LTC-14 cells treated with 100 pM TGF-β for 0, 3 or 6h. ANOVA ***P = 0.0002, Dunnett’s multiple comparison test, 0h versus 3h **P = 0.003, 0h versus 6h ***P = 0.0001. n = 3 replicates. (G) Western blot analyzing downstream TGF-β signaling. LTC-14 cells were serum starved in 1% FBS then treated with 100 pM TGF-β1 ligand for indicated times. (H) Western blot of CYR61 (Abcam) in LTC-14 cells pretreated with DMSO vehicle control or inhibitors against ALK5 (20 μM SB431542), p38 MAPK (10 µM SB203580) or PI3K (10 µM LY294002) for 30min then treated with 100 pM TGF-β1 for 16h. (I) Western blot for CYR61 (Abcam), Smad2 and Smad3 in LTC-14 cells stably expressing NTC or CRISPR constructs against both Smad2 and Smad3. All western blotting results are representative of three independent experiments.

Article Snippet: Antibodies against cleaved caspase 3 (9664), P-Smad2 (3101), Total Smad2 (3103), P-p38 MAPK (4511), Total p38 MAPK (9212), P-Akt (4058), Total Akt (4691) and Total Smad3 (9523) were all purchased from Cell Signaling Technology.

Techniques: Expressing, Microarray, Western Blot, Quantitative RT-PCR, Stable Transfection, CRISPR, Construct

Journal: Cell metabolism

Article Title: Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

doi: 10.1016/j.cmet.2019.01.005

Figure Lengend Snippet: (A) Area-proportional Venn diagram representing the 26 genes significantly (p < 0.05) upregulated both in ob/ob (GSE16790) and db/db (GSE36875) mouse hearts, but not in streptozotocin-induced diabetic hearts (Type I DM) (GSE5606). (B) Pie chart illustrating the percent composition of Gene Ontology biological processes of the 26 common genes found in (A). Metabolic process (GO: 0008152) contains the largest gene set (13 genes), among which only GSK-3α is a kinase. (C) Immunoblots to evaluate nuclear GSK-3α activity in the hearts of wild-type (WT) mice fed a HFD or normal chow (NC) for the indicated periods. GSK-3α was immunoprecipitated from the nuclear fraction of heart lysates, followed by in vitro kinase assays with recombinant β-catenin. Recombinant GSK-3α protein was used as a positive control and immunoprecipitation with IgG was used as a negative control. (D) Quantification of the nuclear GSK-3α activity in (C) (n = 3). (E to L) GSK-3α cardiac-specific heterozygous knockout (GSK-3α cHKO) mice and heterozygous floxed (control) mice were fed a HFD or NC for 14 weeks. (E) Photograph of the hearts of control and GSK-3α cHKO mice fed a HFD or NC. (F) Left ventricular (LV) weight normalized by tibia length, a marker of cardiac hypertrophy (n = 8 (NC) and 22–24 (HFD)). (G and H) Diastolic function, as indicated by deceleration time (n = 8–15) (G), and the slope of the end-diastolic pressure-volume (PV) relation (EDPVR) (n = 5 (NC) and 9 (HFD)) (H). (I) Lipid accumulation in the hearts (Oil Red O staining, left). Scale bar, 100 μm. Inset scale bar, 20 μm. Quantification of myocardial lipid accumulation (right) (n = 6). (J) Palmitate oxidation in the hearts (n = 8–9 (NC) and 17 (HFD)). (K) Picric acid sirius red (PASR) staining, indicating cardiac fibrosis (left). Scale bar, 100 μm. Percentage of PASR positive areas (right) (n = 4). (L) mRNA expression related to cardiac metabolism, inflammation, and transcription factors in the hearts (n = 6). (M) Gene set enrichment analysis plot of Kyoto encyclopedia of genes and genomes (KEGG). PPAR signaling signatures in GSK-3α S21A knock-in (KI) and WT mice fed NC. NES denotes normalized enrichment score. FDR denotes false discovery rate. Error bars indicate s.e.m. * p<0.05, ** p<0.001. See also Figures S1 and S2 and Table S1.

Article Snippet: The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich).

Techniques: Western Blot, Activity Assay, Immunoprecipitation, In Vitro, Recombinant, Positive Control, Negative Control, Knock-Out, Marker, Staining, Expressing, Knock-In

Journal: Cell metabolism

Article Title: Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

doi: 10.1016/j.cmet.2019.01.005

Figure Lengend Snippet: (A) Immunoblots examining the effect of fenofibrate, a PPARα agonist, on PPARα-Ser280 phosphorylation in cardiomyocytes (CMs) in the presence or absence of 500 μM of BSA-palmitic acid (PA). (B to E) Wild-type (WT) mice were fed a high-fat diet (HFD) in the presence or absence of fenofibrate (Feno) for the indicated periods, as shown in Figure S7C. (B) Immunoblots examining the effect of fenofibrate on PPARα-Ser280 phosphorylation in the hearts. (C) Left ventricular (LV) weight normalized by tibia length (n = 8–12). (D) The slope of the end-diastolic pressure-volume (PV) relation (EDPVR), a marker of diastolic function (n = 5–11). (E) Oil Red O staining of heart sections after 14 weeks of HFD in the presence or absence of fenofibrate (left). Scale bar, 100 μm. Quantification of myocardial lipid accumulation (right) (n = 5). (F) The differential effect of fenofibrate on PPRE-luciferase reporter activity in H9C2 cells transduced with PPARα-WT or PPARα-S280D mutant in the presence of a high concentration of fatty acid (500 μM of PA) or BSA control. YFP alone was used for background extraction (n = 6). (G) Fatty acid oxidation (FAO) rate in CMs in the presence of 50 μM or 500 μM of PA. Total oxygen consumption rate (OCR) in CMs transduced with the indicated adenoviruses was measured by 96-well Seahorse experiment and mitochondrial FAO was evaluated by etomoxir-inhibitable OCR (n = 18–23 (WT) and 6–7 (S280D)). (H) Oil Red O staining of rat neonatal CMs transduced with the indicated adenovirus in the presence or absence of fenofibrate with 50 μM or 500 μM of BSA-PA (left). Scale bar, 50 μm. Quantification of myocardial lipid accumulation (right) (n = 6). (I and J) Immunoprecipitation assays showing the interaction between endogenous GSK-3α and YFP-PPARα or YFP alone as a control in CMs treated with 100 μM or 500 μM of BSA-PA in the presence or absence of 10 μM of fenofibrate (I) and quantification of the data (n = 4) (J). (K) Proximity Ligation Assay (PLA) showing the in situ interaction between endogenous GSK-3α and YFP-PPARα in CMs treated with 100 μM or 500 μM of PA in the presence or absence of 10 μM of fenofibrate. PLA was performed using anti-GSK-3α and anti-GFP-YFP antibodies. Red color indicates localization in close proximity. Scale bar, 10 μm. (L) Immunoblots examining GSK-3α activity in failing human hearts in the presence (n = 7) or absence (n = 14) of diabetes. See also Figure S7 and Table S4.

Article Snippet: The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich).

Techniques: Western Blot, Marker, Staining, Luciferase, Activity Assay, Transduction, Mutagenesis, Concentration Assay, Immunoprecipitation, Proximity Ligation Assay, In Situ

Journal: Cell metabolism

Article Title: Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

doi: 10.1016/j.cmet.2019.01.005

Figure Lengend Snippet: Top 10 upregulated KEGG gene sets in GSK-3α KI (vs WT) mouse heart.

Article Snippet: The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich).

Techniques:

Journal: Cell metabolism

Article Title: Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

doi: 10.1016/j.cmet.2019.01.005

Figure Lengend Snippet: (A and B) Immunoprecipitation assays to test the interaction between endogenous GSK-3α and exogenously expressed PPARα. YFP-tagged PPARα or FLAG-tagged PPARα was overexpressed in CMs using adenovirus (A) or in transgenic mouse hearts under the control of the αMHC-promoter (B), respectively. (C) Co-immunoprecipitation assays testing the interaction between endogenous GSK-3α and endogenous PPARα in CMs. (D) In vitro binding assays testing the direct interaction between recombinant (r) GSK-3α and rPPARα. (E to G) Immunoprecipitation assays to identify the amino acids in PPARα responsible for the interaction with endogenous GSK-3α. (E) Schematic representation of rGST-fused PPARα fragments. (F) Schema of the immunoprecipitation assays. rGST-fused-PPARα-full length (FL) or truncated PPARα (T1 to 5) was incubated with lysates extracted from cultured CMs, followed by pull-down with glutathione-sepharose and immunoblotting with anti-GSK-3α antibody. (G) Coomassie Brilliant Blue staining of rGST-PPARα-FL or truncated rGST-PPARα (T1 to T5) (left). Immunoblots testing the binding of endogenous GSK-3α to rGST-PPARα-FL or T1 to T5 (right). (H) Mass spectrometry analysis of the rGST-PPARα protein phosphorylated by GSK-3α in a kinase reaction. The MS/MS spectrum of the PPARα residue corresponding to Ser280 was increased at 80 Da, indicating phosphorylation. (I) Immunoblots showing Ser280 phosphorylation of endogenous PPARα in the hearts of WT mice fed a high-fat diet (HFD) or normal chow (NC) for 3 weeks. α-sarcomeric actinin was used as a loading control. (J) Immunoblots showing pPPARα (S280) in the hearts of control or GSK-3α cHKO mice fed a HFD for the indicated period. See also Figure S3.

Article Snippet: The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich).

Techniques: Immunoprecipitation, Transgenic Assay, In Vitro, Binding Assay, Recombinant, Incubation, Cell Culture, Western Blot, Staining, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: Cell metabolism

Article Title: Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

doi: 10.1016/j.cmet.2019.01.005

Figure Lengend Snippet: (A) Immunoblots showing subcellular localization and expression of p-PPARα (Ser280) in cultured cardiomyocytes (CMs) treated with BSA-palmitic acid (PA) (0–500 μM) for 9 hours. (B) Immunoblots examining the involvement of GSK-3α in the BSA-PA-induced increase in PPARα phosphorylation at Ser280 in CMs. CMs transduced with adenovirus harboring shRNA-GSK-3α or scramble were treated with the indicated concentrations of PA, followed by nuclear extraction and immunoblotting. (C) Immunoblots showing the expression of p-PPARα (Ser280) in the nucleus of CMs treated with BSA-fatty acid cocktail for 9 hours. (D) Clustergram heat map of RNA-sequencing data. H9C2 cells were transduced with PPARα-wild type (WT), -S280A (SA) or -S280D (SD) mutant, or YFP alone as a control. Gene sets having 1) a fold difference of 1.5 or more between SA and SD and 2) a WT expression level located between SD and SA are shown in the heat map. (E) Gene set enrichment analysis plots of PPARα-SD (vs WT) showing upregulated signatures related to energy metabolism. Gene expression was determined by RNA-seq data. (F) qRT-PCR validation of expression of genes related to lipid metabolism and oxidative phosphorylation in CMs transduced with adenovirus harboring a YFP-PPARα-S280A or -S280D mutant or YFP alone as a control (n = 8–10). (G) Fatty acid uptake into CMs transduced with the indicated adenovirus. 3H-palmitate (left) and combined 3H-palmitate and 3H-oleate (right) incorporation into CMs was measured by scintillation counting (n = 4–5). (H) Relative oxygen consumption rate (OCR) of CMs transduced with the indicated adenovirus was measured in a 24-well Seahorse experiment in the presence of 500 μM of BSA-PA. Mitochondrial fatty acid oxidation (FAO) was evaluated by etomoxir-inhibitable OCR. Histograms show FAO rate (the ratio of FAO versus total OCR) (n = 5). (I) FAO rate in CMs transduced with the indicated adenovirus, measured in a 96-well Seahorse experiment in the presence of 500 μM of BSA-fatty acid cocktail (n = 8). (J and K) Oil Red O staining of CMs transduced with the indicated adenovirus in the presence or absence of BSA-PA (500 μM) (n = 5) (J) or in the presence of BSA-fatty acid cocktail (500 μM) (n = 6) (K). BSA alone was used as a control (PA 0 μM). Scale bars, 100 μm (upper panel) and 20 μm (lower panel). Error bars indicate s.e.m. * p < 0.05, ** p < 0.001. See also Figure S4 and Table S2.

Article Snippet: The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich).

Techniques: Western Blot, Expressing, Cell Culture, Transduction, shRNA, RNA Sequencing Assay, Mutagenesis, Quantitative RT-PCR, Staining

Journal: Cell metabolism

Article Title: Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

doi: 10.1016/j.cmet.2019.01.005

Figure Lengend Snippet: (A) Representative immunoblots showing the interaction between YFP-PPARα-wild type (WT), -S280D (SD) or -S280A (SA) mutant and RXRα in cardiomyocytes (CMs) in vitro. YFP alone was used as a control. (B) PPRE-luciferase reporter assay using a series of alanine mutations to evaluate the effect of the indicated basic residues on the activity of PPARα-S280D (n = 12). ## p<0.001 compared to PPARα-S280D. (C) Chromatin immunoprecipitation (ChIP) assays using CMs transduced with adenovirus (Ad)-YFP-PPARα-WT, -SD, -SA, or YFP alone as a control. DNA was amplified by PCR with specific primers flanking the promoter of the indicated genes containing the PPARα-binding motif. PCR using input DNA as template served as an internal control. The data shown are representative of three independent experiments. (D) ChIP assays using specific primers flanking the promoter of the indicated genes containing the PPARα-binding motif. The data shown are representative of three independent experiments. (E) Double-stranded oligo pull-down assays, using biotinylated oligos containing the specific PPRE sequences in the indicated gene promoters. Recombinant GST-PPARα-WT or -SA was subjected to in vitro kinase assays using recombinant GSK-3α prior to the oligo pull-down assays. An oligo containing a PPRE with mutations in four base pairs was used as a negative control. (F) Venn diagram showing the number of genes in the rat genome containing the specific PPRE/DR1 motif (shown in Figure S5F) in their promoters. The numbers of overlapping genes in the Venn diagram (red circle) were significantly different (p=0.0002, Fisher’s exact test). See also Figure S5.

Article Snippet: The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich).

Techniques: Western Blot, Mutagenesis, In Vitro, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, Transduction, Amplification, Binding Assay, Recombinant, Negative Control

Journal: Cell metabolism

Article Title: Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

doi: 10.1016/j.cmet.2019.01.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich).

Techniques: Subcloning, Recombinant, Transfection, Protease Inhibitor, Luciferase, In Situ, Plasmid Preparation, Purification, Gel Extraction, Knock-In, Microarray, Transgenic Assay, Cell Culture, shRNA, Software

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Western blot analysis 96h after Dox-induced shRNA-mediated SOX6 knockdown in RDES and TC-32 EwS cells. GAPDH served as loading control. b) Top: Volcano plot of microarray data showing differentially expressed genes (DEGs) after shRNA-mediated SOX6 knockdown compared to a non-targeting shCtrl. A summary of two EwS cell lines is shown. Bottom: Representative enrichment plots from GSEA of transcriptome profiles of RDES and TC-32 EwS cells 96h after induction of shRNA-mediated SOX6 silencing. c) Left: Quantification of the sphere index after 12 days of Dox-treatment in RDES and TC-32 cells. Horizontal bars represent means and whiskers the SEM, n =3. P values determined via two-sided Mann-Whitney test. Right: Representative micrographs of RDES/TR/shSOX6_3 spheres. Scale bar=1 mm. d) Analysis of tumor growth of xenografted RDES and TC-32 cells containing either Dox-inducible specific shRNAs against SOX6 (shSOX6_2/shSOX6_3) or a non-targeting control shRNA (shCtrl). When tumors were palpable (arrow), mice were randomized and henceforth treated with Dox (+) or vehicle (–). Data are represented as means and SEM, n ≥3 mice per condition. P values determined via two-sided Mann-Whitney test. e) Representative micrographs of xenografts from ( d ) showing IHC stains for SOX6, cleaved caspase 3 and Ki67. Scale bar=20µm. f) Quantification of the relative number of mitoses per high-power field (HPF) of xenografts shown in ( d ). Horizontal bars represent means and whiskers the SEM, n ≥3. P values determined via two-sided Mann-Whitney test. g) Quantification of the relative number of cells positive for cleaved caspase 3 of xenografts shown in ( d ). Horizontal bars represent means and whiskers the SEM, n ≥3. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: Slides were incubated with the polyclonal cleaved caspase 3 primary antibody (rabbit, 1:100; 9661, Cell Signaling, Frankfurt am Main, Germany) for 60 min at RT followed by ImmPRESS Reagent Kit.

Techniques: Western Blot, shRNA, Microarray, MANN-WHITNEY

Journal: bioRxiv

Article Title: Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

doi: 10.1101/578666

Figure Lengend Snippet: a) Analysis of publicly available matched gene expression and drug-response data of up to 22 EwS cell lines per drug. Highlighted in dark grey, top 7 drugs with P <0.02; pink = Elesclomol. b) LN_IC50 (µM) of the top 7 drugs including Federatinib (JAK-2 inhibitor), PHA-793887 (CDK2/5/7 inhibitor), Rucaparib (PARP inhibitor), Serdemetan (p53 activator), Imatinib (tyrosine kinase inhibitor) and Olaparib (PARP1/2 inhibitor) with P <0.02. Horizontal bars represent means and whiskers SEM, n ≥18 EwS cell lines. c ) Quantification of relative viability of indicated cell lines by a Resazurin assay after treatment with Elesclomol at indicated concentrations for 72h. Modeled dose-response curves and calculated IC50 values (nM) are displayed for SOX6-high expressing EwS cells (black and grey) and the SOX6-low expressing osteosarcoma cell line SAOS-2 and the mesenchymal cell line MSC-52 (dark and light green), n ≥3. d ) Analysis of relative SOX6 expression in indicated cell lines by qRT-PCR. Horizontal bars represent means and whiskers SEM, n ≥3. P values determined via two-sided Mann-Whitney test. e ) Analysis of cell viability of indicated cell lines by a Resazurin assay. Horizontal bars represent means and whiskers SEM, n ≥5. P values determined via two-sided Mann-Whitney test. f ) Quantification of relative Elesclomol IC50 values by a Resazurin assay in indicated cell lines after 72h of Elesclomol treatment and concomitant addition of Dox. Horizontal bars represent means and whiskers SEM, n =7. P values determined via two-sided Mann-Whitney test. g ) Quantification of relative Annexin V positivity of indicated EwS cells 48h after treatment with Elesclomol (10 nM). Horizontal bars represent means and whiskers SEM, n =10. P values determined via unpaired two-sided t-test with Welch’s correction. h ) Analysis of tumor growth of TC-32 EwS cells in NSG mice treated once per day (day 0-4 and day 7-9) with Elesclomol (intravenously, 5 mg/kg). Data represent means and SEM, n =5 mice per condition. P values determined via two-sided Mann-Whitney test. i ) Left: Quantification of the average number of cleaved caspase 3 positive cells per 3 HPF in TC-32 xenografts shown in ( h ). Horizontal bars represent means and whiskers SEM, n =5 per condition. P values determined via two-sided Mann-Whitney test. Right: representative micrographs. Scale bar=100 µm. j ) Left: Quantification of necrotic area in TC-32 xenografts shown in ( h ). Horizontal bars represent means and whiskers SEM, n =5 per condition. P values determined via two-sided Mann-Whitney test. Right: Representative micrographs. Scale bar = 900 µm. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: Slides were incubated with the polyclonal cleaved caspase 3 primary antibody (rabbit, 1:100; 9661, Cell Signaling, Frankfurt am Main, Germany) for 60 min at RT followed by ImmPRESS Reagent Kit.

Techniques: Expressing, Resazurin Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: eLife

Article Title: Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

doi: 10.7554/eLife.17047

Figure Lengend Snippet: Transcriptional and biochemical analyses were performed on Cdh16 Cre and Cdh16 Cre ::Tfeb fs mice. ( A , B ) Tables show the relative increase of genes related to the ErbB ( A ) and WNT ( B ) pathways in the microarray analyses performed on kidneys from P0 Cdh16 Cre ::Tfeb fs mice. Graphs show real-time PCR validations performed on kidneys from Cdh16 Cre ::Tfeb fs mice at different stages (P0, P12, P30). Data are shown as the average (± SEM) of at least three Cdh16 Cre ::Tfeb fs mice normalized versus wild-type mice. ( C , D ) Immunoblot analyses performed on ( C ) P30 kidney tissues and ( D ) primary kidney cells isolated from Cdh16 Cre ::Tfeb fs mice to evaluate ErbB and WNT activation status. Each replicate is a distinct biological sample. ErbB signaling was assessed by looking at phosphoAKT (Ser473) to total AKT ratio, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK ratio; WNT signaling was assessed by quantifying β-catenin and CCND1 (Cyclin D1) protein levels. Graphs represent the densitometry quantification of Western blot bands. Values are normalized to actin when not specified and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test). DOI: http://dx.doi.org/10.7554/eLife.17047.007 10.7554/eLife.17047.008 Figure 3—source data 1. Complete list of 294 genes (represented by 361 probesets) significantly induced (FDR≤0.05) in the KSP_P0 microarray dataset (GSE62977). The genes are ranked by decreasing signed ratio (KSP_P0/CTL). DOI: http://dx.doi.org/10.7554/eLife.17047.008 10.7554/eLife.17047.009 Figure 3—source data 2. Complete list of 628 genes (represented by 729 probesets) significantly induced (FDR≤0.05) in the KSP_P14 microarray dataset (GSE63376). The genes are ranked by decreasing signed ratio (KSP_P14/CTL). DOI: http://dx.doi.org/10.7554/eLife.17047.009

Article Snippet: For Western blots, the following antibodies were used: anti-FLAG M2-HRP (Sigma, cat. A8592, RRID: AB_439702 , 1:1000), anti-actin (Sigma, cat. A2066, RRID: AB_476693 , 1:5000), anti-βtubulin (Sigma, cat. T8328, RRID: AB_1844090 1:1000), anti-Human/Mouse/Rat Pan-Akt (R&D, cat. MAB2055, RRID: AB_2224581 , 1:500), Phospho-Akt (Ser473) (D9E) Cell Signaling, cat. #4060, RRID: AB_2315049 , 1:1000), anti-human, mouse, and rat ERK1/ERK2 (R&D, cat.216703, RRID: AB_2140121 , 1:2000), anti-Human/Mouse/Rat Phospho- ERK1(T202/Y204)/ERK2 (T185/Y187) (R&D, cat. AF1018, RRID: AB_354539 1:1000), anti-β-catenin (BD, cat. 610154, RRID: AB_397555 1:500), anti-active β-catenin (Cell Signaling, cat. #8814, RRID: AB_11127203 1:1000), anti-Cyclin D1 (Cell Signaling, cat. #2978, RRID: AB_10692801 1:1000), anti-LRP6 (Cell Signaling, cat. #3395, RRID: AB_1950408 1:1000), anti-phospho-LRP6 (Ser1490) (Cell Signaling, cat. #2568, RRID: AB_2139327 1:1000), anti-GSK3β (Cell Signaling, cat. #9315, RRID: AB_490890 1:1000), anti-phospho-GSK3β (Ser9) (Cell Signaling, cat. #9323, RRID: AB_2115201 1:1000), anti MYC (Cell Signaling, cat. #5605, RRID: AB_1903938 1:1000).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Western Blot, Isolation, Activation Assay

Journal: eLife

Article Title: Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

doi: 10.7554/eLife.17047

Figure Lengend Snippet: Immunoblot analysis performed on P90 kidneys from Cdh16 Cre ::Tfeb fs mice ( A ) and P90 Cdh16 CreErt2 ::Tfeb fs animals induced with tamoxifen at P14 ( B ) and at P30 ( C ), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test). DOI: http://dx.doi.org/10.7554/eLife.17047.011

Article Snippet: For Western blots, the following antibodies were used: anti-FLAG M2-HRP (Sigma, cat. A8592, RRID: AB_439702 , 1:1000), anti-actin (Sigma, cat. A2066, RRID: AB_476693 , 1:5000), anti-βtubulin (Sigma, cat. T8328, RRID: AB_1844090 1:1000), anti-Human/Mouse/Rat Pan-Akt (R&D, cat. MAB2055, RRID: AB_2224581 , 1:500), Phospho-Akt (Ser473) (D9E) Cell Signaling, cat. #4060, RRID: AB_2315049 , 1:1000), anti-human, mouse, and rat ERK1/ERK2 (R&D, cat.216703, RRID: AB_2140121 , 1:2000), anti-Human/Mouse/Rat Phospho- ERK1(T202/Y204)/ERK2 (T185/Y187) (R&D, cat. AF1018, RRID: AB_354539 1:1000), anti-β-catenin (BD, cat. 610154, RRID: AB_397555 1:500), anti-active β-catenin (Cell Signaling, cat. #8814, RRID: AB_11127203 1:1000), anti-Cyclin D1 (Cell Signaling, cat. #2978, RRID: AB_10692801 1:1000), anti-LRP6 (Cell Signaling, cat. #3395, RRID: AB_1950408 1:1000), anti-phospho-LRP6 (Ser1490) (Cell Signaling, cat. #2568, RRID: AB_2139327 1:1000), anti-GSK3β (Cell Signaling, cat. #9315, RRID: AB_490890 1:1000), anti-phospho-GSK3β (Ser9) (Cell Signaling, cat. #9323, RRID: AB_2115201 1:1000), anti MYC (Cell Signaling, cat. #5605, RRID: AB_1903938 1:1000).

Techniques: Western Blot