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: 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: 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: 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: 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: 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: 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: bioRxiv

Article Title: Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

doi: 10.1101/623975

Figure Lengend Snippet: ( A ) Venn diagrams showing the overlap of H3K9me3 and H3K14ac marks in young (left panel) and old (right panel) livers, identifying 1032 and 668 bivalent regions (H3K9me3/K14ac) with 280 domains were present at both conditions (middle panel). ( B ) Examples of genomic regions with H3K9me3/H3K14ac bivalent mark specific to young liver (left panel: chr2:177,312,993-177,319,994, bivalent region in red rectangle), common to both (middle panel: chr12:67,058,502-67,060,498). and old livers (right panel: chr13:65,257,743-65,261,994, bivalent region in red rectangle). Magnitude of the ChIP-Seq signal is shown on y-axis. For comparison, tracks of a given mark in young and old conditions are group scaled and input track is set to the lowest of the magnitudes in the view. ( C ) Heatmaps showing H3K9me3 and H3K14ac ChIP-Seq signal at bivalent regions specific to young (left panel), common to both (middle panel), and specific to old livers (right panel). ( D ) Comparison of over-represented disease functions (top bar graph) and pathways (bottom bar graph) identified by Ingenuity Pathway Analysis of genes associated with bivalent mark in young livers (Young BM black bar) and differentially regulated genes in aged livers (DE Old vs. Young, red bar). Genes associated with bivalent regions are identified by Genomic Regions Enrichment of Annotations Tool (GREAT, see methods for details). ( E ) Single gene perturbation analysis by Enrichr calculates statistical significance of the overlap of a gene set differentially expressed in a knockout of each factor and the input gene set. Analysis of enriched gene sets in genes associated with bivalent mark in young livers (Young BM) identified Sirt1 target genes from livers Sirt1 KO mice as the most significantly enriched ( p -value 2.28×10 −07 ) among Young BM. ( F ) Chromatin-x Enrichment Analysis (ChEA 2016) of bivalent regions of young (Young BM, black bar) and old (Old BM, gray bar) livers by Enrichr shows Kap1 ( p -value young: 2.89×10 −11 , old: 7.55×10 −10 ) binding sites are most significantly enriched at these bivalent regions. Reads are merged from two replicates in each condition. ChIP-Seq data for H3K9me3 and RNA-Seq data for differentially regulated genes in aged liver are from our previous studies.

Article Snippet: Rabbit monoclonal antibody for Sirt1 (CST, 9475) and rabbit polyclonal antibodies specific to KAP1 (Abcam, ab10483) and SETDB1 (Proteintech, 11231-1-AP) were used in western blotting at 1:1000 dilution.

Techniques: ChIP-sequencing, Knock-Out, Binding Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

doi: 10.1101/623975

Figure Lengend Snippet: ( A ) Relative mRNA levels (n=4, left panel) of Sirt1 by quantitative RT-PCR. Gapdh was used as a housekeeping gene. Sirt1 mRNA levels do not change in old livers. Western blot analysis (n=3, right panel) of protein nuclear extracts with antibodies to Sirt1 and Lmnb1 (loading control) in young and old livers. Protein levels of Sirt1 are decreased in old livers. ( B ) Model implicating the histone deacetylase, Sirt1, as a key regulator of H3K9me3/K14ac bivalent mark (BM). Sirt1-mediated deacetylation of H3K14ac leads to loss of bivalent mark (no BM, top panel) and loss of Sirt1 activity allows for formation of new domains (BM, bottom panel). In each case, heatmaps show the corresponding changes in the H3K14ac signal. ( C ) Comparison of overrepresented disease functions (top panel) and pathways (bottom panel) identified by Ingenuity Pathway Analysis between differentially expressed genes in aged liver (DE Young vs. Old, red bar) and Sirt1-regulated genes associated with young liver bivalent mark (Sirt1-regulated Young BM, black bar). ( D ) Analysis of over represented pathways in Sirt1-regulated genes associated with old liver-specific bivalent regions by Enrichr (Sirt1-regulated Old BM, gray bar). Formation of senescence associated heterochromatin ( p -value 8.56×10 −5 ) was most significantly enriched pathway. Data set for differentially regulated genes in aged livers is an RNA-Seq data set from our previous study and for Sirt1-regulated genes is from a published microarray study (Purushotam et al., 2009).

Article Snippet: Rabbit monoclonal antibody for Sirt1 (CST, 9475) and rabbit polyclonal antibodies specific to KAP1 (Abcam, ab10483) and SETDB1 (Proteintech, 11231-1-AP) were used in western blotting at 1:1000 dilution.

Techniques: Quantitative RT-PCR, Western Blot, Histone Deacetylase Assay, Activity Assay, RNA Sequencing Assay, Microarray

Journal: bioRxiv

Article Title: Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

doi: 10.1101/623975

Figure Lengend Snippet: In the first step, close residues H3K9 and H3K14 are acetylated by the same histone acetyltransferase (HAT). Then, H3K9 is deacetylated by Hdac3, allowing for subsequent triple methylation of H3K9 by Setdb1, which is found in complex with Kap1, and establishment of the bivalent region. Sirt1 activity is inhibited during bivalent region assembly to preserve acetylation of H3K14 that serves as the first step in that process. Activation of Sirt1 activity leads to loss of the bivalent mark.

Article Snippet: Rabbit monoclonal antibody for Sirt1 (CST, 9475) and rabbit polyclonal antibodies specific to KAP1 (Abcam, ab10483) and SETDB1 (Proteintech, 11231-1-AP) were used in western blotting at 1:1000 dilution.

Techniques: Methylation, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: RUNX1 suppresses breast cancer stemness and tumor growth

doi: 10.1101/315093

Figure Lengend Snippet: (A) Representative tissue microarray images of RUNX1 in normal breast tissue. (B) and (C) Representative tissue microarray images of RUNX1 in breast tumor tissues. (D) Kaplan-Meier analysis showed higher overall survival in patients with higher RUNX1 mRNA expression (GSE37751, GSE7390 and TCGA). Gehan-Breslow-Wilcoxon test with p value<0.01, p value<0.05, p value<0.01 respectively compared with high RUNX1 expression patients and low RUNX1 expression patients in three data sets.

Article Snippet: Detection was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336), a mouse monoclonal CD24 (Santa-Cruz sc-11406).

Techniques: Microarray, Expressing

Journal: bioRxiv

Article Title: RUNX1 suppresses breast cancer stemness and tumor growth

doi: 10.1101/315093

Figure Lengend Snippet: (A) MCF10CA1a cells were injected into the mammary fat pad of SCID mice. Points represent mean tumor volume. (B) Western blot analyses show RUNX1 and E-cadherin levels are decreased and Vimentin level is increased in tumor samples compared to MCF10CA1a cells. (C) Upper panel, Protein quantification show that RUNX1 is significant decreased in tumor samples compared to MCF10CA1a. Data shown represent mean ± SEM from three independent experiments. Lower panel, RT-qPCR analyses of RNA from tumor samples show decreased RUNX1 expression of compared with MCF10CA1a cells. Student’s t test * p value <0.05, *** p value <0.001 and. Error bars represent the standard error of the mean (SEM) from three independent experiments. (D) RT-qPCR analyses of RNA from tumor samples show activation of mesenchymal marks Vimentin and FN1 and other tumor growth related genes including MMP9, MMP13, VGF, CXCR4 and CXCL12 compared with MCF10CA1a cells. Student’s t test * p value <0.05, ** p value <0.01, *** p value <0.001 and **** p value <0.0001. Error bars represent the standard error of the mean (SEM) from three independent experiments.

Article Snippet: Detection was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336), a mouse monoclonal CD24 (Santa-Cruz sc-11406).

Techniques: Injection, Western Blot, Quantitative RT-PCR, Expressing, Activation Assay

Journal: bioRxiv

Article Title: RUNX1 suppresses breast cancer stemness and tumor growth

doi: 10.1101/315093

Figure Lengend Snippet: (A) Western blot analyses confirm RUNX1 overexpression in MCF10CA1a (Upper) and MCF10AT1 (Lower) cells. Vimentin expression is repressed upon RUNX1 overexpression in both cell lines. (B) Representative phase contrast images (magnification 100×) of MCF10AT1 and MCF10CA1a cells with EV control or RUNX1 overexpression subjected to a scratch assay for times indicated. The area of the scratch was plotted as a percentage of total area for N = 3 independent experiments carried out in duplicate. Data shown represent mean ± SEM from three independent experiments using student t-test. (C) Light microscopy images (mag. 12×) of stained cells from a representative (1 of N = 2) trans-well migration assay experiment MCF10AT1 and MCF10CA1a cells with EV control or RUNX1 overexpression ( left ); quantitation of migrated cells assessed by measurement of the absorbance of solubilized crystal violet stain retained by migrated cells ( right ). (D) Light microscopy images (mag. 12×) of stained cells from a representative (1 of N = 2) trans-well matrigel invasion assay experiment with MCF10AT1 and MCF10CA1a cells with EV control or RUNX1 overexpression to evaluate invasion ( left ); quantitation of invaded cells assessed by measurement of the absorbance of solubilized crystal violet stain retained by invaded cells ( right ). For all assays, three independent experiments were carried out in duplicates. All quantitative data are depicted as mean ± S.E.M per group. * P < 0.05, ** P < 0.01 (student’s t -test).

Article Snippet: Detection was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336), a mouse monoclonal CD24 (Santa-Cruz sc-11406).

Techniques: Western Blot, Over Expression, Expressing, Wound Healing Assay, Light Microscopy, Staining, Migration, Quantitation Assay, Invasion Assay

Journal: bioRxiv

Article Title: RUNX1 suppresses breast cancer stemness and tumor growth

doi: 10.1101/315093

Figure Lengend Snippet: (A) A total of 1 × 10 6 MCF10CA cells with EV or RUNX1 overexpression were injected into mammary fat pad of SCID mice ( n = 7 in each group). The points represent average tumor volume at each time point ± S.E.M. P values were obtained by 2-tailed Student t test. * , P < 0.05; *** , P<0.001; **** , P<0.0001. (B) Tumor size measured at day 28 (end point). P values were obtained by 2-tailed Student t test. * , P < 0.05. (C) Tumor weight at day 28 (end point). P values were obtained by 2-tailed Student t test. * , P < 0.05. (D) Representative luminescence images at 4 weeks after mammary fat pad injection.

Article Snippet: Detection was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336), a mouse monoclonal CD24 (Santa-Cruz sc-11406).

Techniques: Over Expression, Injection

Journal: bioRxiv

Article Title: RUNX1 suppresses breast cancer stemness and tumor growth

doi: 10.1101/315093

Figure Lengend Snippet: (A) Western blot analyses show RUNX1 is decreased and Zeb1, Twist1 and Vimentin level are increased in BCSC samples compared to Parental and Bulk MCF10AT1 cells. Right, protein quantification shows that RUNX1 is significant decreased in BCSC. (B) Tumorsphere formation efficiency for BCSC populations is significantly higher than bulk population. Data shown represent mean ± SEM from three independent experiments. ** P < 0.01. (C) RUNX1 overexpression in MCF10CA1a cells reduces tumorsphere formation efficiency. Data shown represent mean ± SEM from three independent experiments. * P < 0.05. Right, represent picture of tumorsphere. (D) RUNX1 overexpression in MCF10AT1 cells reduces tumorsphere formation efficiency. Data shown represent mean ± SEM from three independent experiments. * P < 0.05 Right, represent picture of tumorsphere. (E) Western blot analyses of lysates from MCF10AT1 cells treated with shRUNX1 show decreased protein expression of RUNX1 and E-cadherin and increased protein expression of Vimentin. (F) RUNX1 knockdown in MCF10AT1 cells activates tumorsphere formation efficiency. Data shown represent mean ± SEM from three independent experiments. * P < 0.05. Right, represents picture of tumorsphere.

Article Snippet: Detection was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336), a mouse monoclonal CD24 (Santa-Cruz sc-11406).

Techniques: Western Blot, Over Expression, Expressing

Journal: bioRxiv

Article Title: RUNX1 suppresses breast cancer stemness and tumor growth

doi: 10.1101/315093

Figure Lengend Snippet: (A) Flow cytometric analysis of CD44 and CD24 expression in MCF10AT1 cells with EV or RUNX1 overexpression. Data shown represent mean ± SEM from three independent experiments. p Values were determined by student t test (B) Flow cytometric analysis of CD44 and CD24 expression in MCF10AT1 cells stably expressing RUNX1 or non-silencing shRNAs. Data shown represent mean ± SEM from three independent experiments. p Values were determined by student t test.

Article Snippet: Detection was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336), a mouse monoclonal CD24 (Santa-Cruz sc-11406).

Techniques: Expressing, Over Expression, Stable Transfection

Journal: bioRxiv

Article Title: RUNX1 suppresses breast cancer stemness and tumor growth

doi: 10.1101/315093

Figure Lengend Snippet: (A) Western blot analyses show Zeb1 is decreased upon RUNX1 overexpression in MCF10AT1 cells. (B) Western blot analyses show Zeb1 is activated upon RUNX1 knockdown in MCF10AT1 cells. (C) ChIP-qPCR confirmation of RUNX1 occupancy at Zeb1. RUNX1 binding is increased in RUNX1 overexpression samples. Data obtained with antibodies against RUNX1 are normalized to input control and ZNF188 (NC1) and ZNF333 (NC2), which were used as the negative control as RUNX1 are predicted not to bind these genes. Data shown represent mean ± SEM from three independent experiments. p Values were determined by student t test. (D) Mechanism on how RUNX1 represses tumor growth in breast cancer. (EC-epithelial like cells; MC-mesenchymal-like cells).

Article Snippet: Detection was performed using a rabbit polyclonal RUNX1 antibody (Cell Signaling #4336), a mouse monoclonal CD24 (Santa-Cruz sc-11406).

Techniques: Western Blot, Over Expression, Binding Assay, Negative Control

Journal: Advanced Science

Article Title: SETDB1 Methylates MCT1 Promoting Tumor Progression by Enhancing the Lactate Shuttle

doi: 10.1002/advs.202301871

Figure Lengend Snippet: SETDB1 interacts with MCT1 and enhances its expression. a,b) Whole cell lysates (WCL) of SW480 and HT29 cells were collected for IP with anti‐MCT1 or anti‐SETDB1 antibody, followed by immunoblots (IB) analysis. c) The co‐localization of MCT1 and SETDB1 was identified by IF analysis using anti‐MCT1 and anti‐SETDB1 antibodies in SW480 cells. White scale bars, 5 µm. d) In vitro binding assay was performed. Purified Flag‐SETDB1 was incubated with His‐MCT1(444‐500aa) and pulled down using anti‐Flag beads, followed by IB analysis. e–h) Protein and mRNA expression of MCT1 were detected by IB assays and qRT‐PCR assays in SW480 and HT29 cells silenced with control (shNC) or SETDB1 shRNA (#1 and #2). i,j) Protein and mRNA expression of MCT1 were measured by IB assays and qRT‐PCR assays in SW480 cells transfected with Vector, Flag‐SETDB1 (WT), and Flag‐SETDB1 (H1224K) plasmids. k,l) Protein and mRNA expression of MCT1 were determined by IB assays and qRT‐PCR assays in SW480 cells treated with Mithramycin A at the indicated concentrations. m) Colorectal tumors and paired normal tissues were extracted and subjected to detection of MCT1 and SETDB1 protein expression by IB analysis. All immunoblots are performed three times, independently, with similar results. f,h,j,l) Data are represented as mean ± s.d. ns means no significant, by one‐way analysis of variance (ANOVA) with Tukey's test.

Article Snippet: The primary antibodies for MCT1 (sc‐365501; Santa Cruz Biotechnology, ab93048; Abcam and A3013; ABclonal), SETDB1 (2196; Cell Signaling Technology and A6145; ABclonal), GAPDH (AC033; ABclonal), Tollip (A21551; ABclonal), ATG5 (10181‐2‐AP; Proteintech), Beclin 1 (11306‐1‐AP; Proteintech), Caveolin‐1 (A19006; ABclonal), Histone H3 (4499; Cell Signaling Technology), trimethyl‐Histone H3‐K9 (A2360; ABclonal), Flag tag (14793S; Cell Signaling Technology), HA tag (51064‐2‐AP; Proteintech), His tag (AE003; ABclonal), Pan mono‐methyl lysine (A18293; ABclonal), Pan di‐methyl lysine (14117; Cell Signaling Technology), Pan tri‐methyl lysine (14680; Cell Signaling Technology), LDHA (A0861; ABclonal), p‐LDHA Y10 (AP0889; ABclonal), Gpr132 (DF4894; Affinity Biosciences), LC3 (3868; Cell Signaling Technology), MRC1 (ab300621; Abcam), and MCT4 (22787‐1‐AP; Proteintech) were commercially purchased.

Techniques: Expressing, Western Blot, In Vitro, Binding Assay, Purification, Incubation, Quantitative RT-PCR, shRNA, Transfection, Plasmid Preparation

Journal: Advanced Science

Article Title: SETDB1 Methylates MCT1 Promoting Tumor Progression by Enhancing the Lactate Shuttle

doi: 10.1002/advs.202301871

Figure Lengend Snippet: SETDB1 represses the autophagic degradation of MCT1. a) The degradation of MCT1 was detected in shSETDB1NC and shSETDB1#1 SW480 cells treated with CHX (100 µg mL −1 ) for 0, 4, 8, or 12 h by CHX‐chase assay. b) Quantification of the relative protein level of MCT1 in (a). c) The protein level of MCT1 was examined in SW480 cells in the presence of different inhibitors MG132 (10 × 10 −6 m ), CQ (50 × 10 −6 m ), or 3‐MA (5 × 10 −3 m ). d) The degradation of MCT1 was evaluated by CHX‐chase assay in ATG5 WT or ATG5 KO SW480 cells. e) Quantification of the relative protein level of MCT1 in (d). f) The degradation of MCT1 was evaluated by CHX‐chase assay in Beclin 1 WT or Beclin 1 KO SW480 cells. g) Quantification of the relative protein level of MCT1 in (f). h,i) The protein level of MCT1 in WT and ATG5 or Beclin 1 KO SW480 cells treated with dimethyl sulfoxide or Mithramycin A (100 × 10 − 9 m , 24 h). All immunoblots were performed three times, independently, with similar results. b,e,g) Data are represented as mean ± s.d. ** p < 0.01, **** p < 0.0001, by two‐way ANOVA with Tukey's test.

Article Snippet: The primary antibodies for MCT1 (sc‐365501; Santa Cruz Biotechnology, ab93048; Abcam and A3013; ABclonal), SETDB1 (2196; Cell Signaling Technology and A6145; ABclonal), GAPDH (AC033; ABclonal), Tollip (A21551; ABclonal), ATG5 (10181‐2‐AP; Proteintech), Beclin 1 (11306‐1‐AP; Proteintech), Caveolin‐1 (A19006; ABclonal), Histone H3 (4499; Cell Signaling Technology), trimethyl‐Histone H3‐K9 (A2360; ABclonal), Flag tag (14793S; Cell Signaling Technology), HA tag (51064‐2‐AP; Proteintech), His tag (AE003; ABclonal), Pan mono‐methyl lysine (A18293; ABclonal), Pan di‐methyl lysine (14117; Cell Signaling Technology), Pan tri‐methyl lysine (14680; Cell Signaling Technology), LDHA (A0861; ABclonal), p‐LDHA Y10 (AP0889; ABclonal), Gpr132 (DF4894; Affinity Biosciences), LC3 (3868; Cell Signaling Technology), MRC1 (ab300621; Abcam), and MCT4 (22787‐1‐AP; Proteintech) were commercially purchased.

Techniques: Western Blot

Journal: Advanced Science

Article Title: SETDB1 Methylates MCT1 Promoting Tumor Progression by Enhancing the Lactate Shuttle

doi: 10.1002/advs.202301871

Figure Lengend Snippet: SETDB1 induces tri‐methylation of lysine 473 on MCT1. a) WCL collected from SW480 and HT29 cells silenced with control (ShNC) or SETDB1 ShRNA (#1, #2) were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. b) WCL collected from SW480 and HT29 cells transfected with Vector, Flag‐SETDB1 (WT), and Flag‐SETDB1 (H1224K) plasmids were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. c) HEK293T cells transfected with HA‐MCT1 WT or mutant plasmids as indicated, then transfected with Vector or Flag‐SETDB1, WCL were collected for IP with anti‐HA beads, followed by IB analysis. d) Secondary mass spectrometry result of lysine 473 methylation residue. e) SW480 MCT1 KO cells transfected with HA‐MCT1 WT or K473R plasmid as indicated, then transfected with Vector or Flag‐SETDB1, WCL were collected for IP with anti‐HA beads, followed by IB analysis. f) Different peptides were added into PVDF membranes at indicated concentrations, followed by a dot blot assay using MCT1 K473 specific tri‐methylation antibody. g) In vitro methylation assay was performed. Purified Flag‐SETDB1 was incubated with His‐MCT1(444‐500aa) in the presence of S‐adenosyl‐L‐methionine, followed by IB analysis to analyze MCT1 methylation using MCT1 K473‐specific tri‐methylation antibody. h) WCL collected from SW480 and HT29 cells silenced with control (ShNC) or SETDB1 ShRNA (#1, #2) were subjected to IB assay with MCT1 K473‐specific tri‐methylation antibody. All immunoblots were performed three times, independently, with similar results.

Article Snippet: The primary antibodies for MCT1 (sc‐365501; Santa Cruz Biotechnology, ab93048; Abcam and A3013; ABclonal), SETDB1 (2196; Cell Signaling Technology and A6145; ABclonal), GAPDH (AC033; ABclonal), Tollip (A21551; ABclonal), ATG5 (10181‐2‐AP; Proteintech), Beclin 1 (11306‐1‐AP; Proteintech), Caveolin‐1 (A19006; ABclonal), Histone H3 (4499; Cell Signaling Technology), trimethyl‐Histone H3‐K9 (A2360; ABclonal), Flag tag (14793S; Cell Signaling Technology), HA tag (51064‐2‐AP; Proteintech), His tag (AE003; ABclonal), Pan mono‐methyl lysine (A18293; ABclonal), Pan di‐methyl lysine (14117; Cell Signaling Technology), Pan tri‐methyl lysine (14680; Cell Signaling Technology), LDHA (A0861; ABclonal), p‐LDHA Y10 (AP0889; ABclonal), Gpr132 (DF4894; Affinity Biosciences), LC3 (3868; Cell Signaling Technology), MRC1 (ab300621; Abcam), and MCT4 (22787‐1‐AP; Proteintech) were commercially purchased.

Techniques: Methylation, shRNA, Transfection, Plasmid Preparation, Mutagenesis, Mass Spectrometry, Residue, Dot Blot, In Vitro, Purification, Incubation, Western Blot

Journal: Advanced Science

Article Title: SETDB1 Methylates MCT1 Promoting Tumor Progression by Enhancing the Lactate Shuttle

doi: 10.1002/advs.202301871

Figure Lengend Snippet: MCT1 K473 tri‐methylation blocks Tollip‐mediated autophagic degradation of MCT1. a) The degradation of MCT1 was detected in SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R cells treated with CHX (100 µg mL −1 ) for 0, 4, 8, or 12 h by CHX‐chase assay. b) Quantification of relative protein level of MCT1 in (a). c) HEK293T cells were transfected with HA‐MCT1 and indicated Flag‐tagged cargo receptors, WCL were collected for IP with anti‐Flag beads, followed by IB analysis. d) The degradation of MCT1 was evaluated by CHX‐chase assay in Tollip WT or Tollip KO SW480 cells. e) Quantification of the relative protein level of MCT1 in (d). f) WCL collected from SETDB1 knockdown SW480 and HT29 cells were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. g) WCL collected from SW480 cells transfected with Vector, Flag‐SETDB1 (WT), and Flag‐SETDB1 (H1224K) plasmids were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. h) HEK293T cells were co‐transfected with Flag‐Tollip and Vector, HA‐MCT1 WT or HA‐MCT1 K473R, and WCL was collected for IP with anti‐HA beads, followed by IB analysis. i) The protein level of MCT1 in Tollip WT and Tollip KO SW480 cells treated with DMSO or Mithramycin A (100 × 10 −9 m , 24 h). All immunoblots were performed three times, independently, with similar results. a,b) Data are represented as mean ± s.d. **** p < 0.0001, by two‐way ANOVA with Tukey's test.

Article Snippet: The primary antibodies for MCT1 (sc‐365501; Santa Cruz Biotechnology, ab93048; Abcam and A3013; ABclonal), SETDB1 (2196; Cell Signaling Technology and A6145; ABclonal), GAPDH (AC033; ABclonal), Tollip (A21551; ABclonal), ATG5 (10181‐2‐AP; Proteintech), Beclin 1 (11306‐1‐AP; Proteintech), Caveolin‐1 (A19006; ABclonal), Histone H3 (4499; Cell Signaling Technology), trimethyl‐Histone H3‐K9 (A2360; ABclonal), Flag tag (14793S; Cell Signaling Technology), HA tag (51064‐2‐AP; Proteintech), His tag (AE003; ABclonal), Pan mono‐methyl lysine (A18293; ABclonal), Pan di‐methyl lysine (14117; Cell Signaling Technology), Pan tri‐methyl lysine (14680; Cell Signaling Technology), LDHA (A0861; ABclonal), p‐LDHA Y10 (AP0889; ABclonal), Gpr132 (DF4894; Affinity Biosciences), LC3 (3868; Cell Signaling Technology), MRC1 (ab300621; Abcam), and MCT4 (22787‐1‐AP; Proteintech) were commercially purchased.

Techniques: Methylation, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: Advanced Science

Article Title: SETDB1 Methylates MCT1 Promoting Tumor Progression by Enhancing the Lactate Shuttle

doi: 10.1002/advs.202301871

Figure Lengend Snippet: MCT1 K473 tri‐methylation is required for SETDB1‐mediated tumor glycolysis and M2‐like polarization of TAMs. a) Lactate production was measured in the conditioned medium (CM) of SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1, n = 3. b) Glucose uptake was measured in SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1, n = 3. c,d) Bioenergetic analysis was performed with the Seahorse XF24 analyzer platform. ECAR of SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1 was measured and calculated, n = 3. e) WCL collected from SW480 MCT1 KO cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1, followed by IB assay. f) The relative mRNA levels of VEGF and ARG1 macrophage markers in BMDMs were treated with indicated CM, n = 3. g) A syngeneic tumor model was performed by injecting MCT1‐3′UTR CT26 cells stably expressing MCT1 WT or MCT1 K473R cells and overexpression Vector or SETDB1 into BALB/c mice. n = 5 mice. h,i) Quantification of tumor weight and volume of tumors generated in (g). j,k) Flow cytometry analysis of macrophage polarization of tumors generated in (g). All immunoblots were performed three times, independently, with similar results. Data are represented as mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns means no significant, by a,b,h,k) one‐way ANOVA with Tukey's test and d,f,i) two‐way ANOVA with Tukey's test.

Article Snippet: The primary antibodies for MCT1 (sc‐365501; Santa Cruz Biotechnology, ab93048; Abcam and A3013; ABclonal), SETDB1 (2196; Cell Signaling Technology and A6145; ABclonal), GAPDH (AC033; ABclonal), Tollip (A21551; ABclonal), ATG5 (10181‐2‐AP; Proteintech), Beclin 1 (11306‐1‐AP; Proteintech), Caveolin‐1 (A19006; ABclonal), Histone H3 (4499; Cell Signaling Technology), trimethyl‐Histone H3‐K9 (A2360; ABclonal), Flag tag (14793S; Cell Signaling Technology), HA tag (51064‐2‐AP; Proteintech), His tag (AE003; ABclonal), Pan mono‐methyl lysine (A18293; ABclonal), Pan di‐methyl lysine (14117; Cell Signaling Technology), Pan tri‐methyl lysine (14680; Cell Signaling Technology), LDHA (A0861; ABclonal), p‐LDHA Y10 (AP0889; ABclonal), Gpr132 (DF4894; Affinity Biosciences), LC3 (3868; Cell Signaling Technology), MRC1 (ab300621; Abcam), and MCT4 (22787‐1‐AP; Proteintech) were commercially purchased.

Techniques: Methylation, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Over Expression, Generated, Flow Cytometry, Western Blot

Journal: Advanced Science

Article Title: SETDB1 Methylates MCT1 Promoting Tumor Progression by Enhancing the Lactate Shuttle

doi: 10.1002/advs.202301871

Figure Lengend Snippet: MCT1 K473 tri‐methylation is positively related to CRC and has prognostic significance in CRC patients. a) MCT1 K473 tri‐methylation IHC staining score was detected in tumor and adjacent tissues, n = 79. Student's two‐tailed t ‐test, p < 0.001. b) Representative image of IHC staining for SETDB1, MCT1 K473 tri‐methylation, and CD206 in colorectal cancer. Black scale bar, 50 µm . c) Scatter plot of the IHC staining scores for SETDB1, MCT1 K473 tri‐methylation, and CD206 in CRC, n = 80. All p and r values were calculated with Spearman's r test. c) Quantitative IHC staining score showing the correlation of SETDB1 and MCT1 K473 tri‐methylation. Chi‐square test, p < 0.0001. d) Correlation between SETDB1 and MCT1 K473 tri‐methylation expression was determined by Pearson correlation coefficient test, p < 0.0001. e) Kaplan–Meier analysis of overall survival in a set of 80 colorectal cancer patients according to MCT1 K473 tri‐methylation expression. Log‐rank test, p = 0.0310. f,g) Quantitative IHC staining score showing the correlation between MCT1 K473 tri‐methylation and TNM stage or N stage using microarray of colorectal cancer specimen. Chi‐square test, p = 0.0155 and p = 0.0352, respectively. h) The working model of SETDB1‐mediated MCT1 K473 tri‐methylation.

Article Snippet: The primary antibodies for MCT1 (sc‐365501; Santa Cruz Biotechnology, ab93048; Abcam and A3013; ABclonal), SETDB1 (2196; Cell Signaling Technology and A6145; ABclonal), GAPDH (AC033; ABclonal), Tollip (A21551; ABclonal), ATG5 (10181‐2‐AP; Proteintech), Beclin 1 (11306‐1‐AP; Proteintech), Caveolin‐1 (A19006; ABclonal), Histone H3 (4499; Cell Signaling Technology), trimethyl‐Histone H3‐K9 (A2360; ABclonal), Flag tag (14793S; Cell Signaling Technology), HA tag (51064‐2‐AP; Proteintech), His tag (AE003; ABclonal), Pan mono‐methyl lysine (A18293; ABclonal), Pan di‐methyl lysine (14117; Cell Signaling Technology), Pan tri‐methyl lysine (14680; Cell Signaling Technology), LDHA (A0861; ABclonal), p‐LDHA Y10 (AP0889; ABclonal), Gpr132 (DF4894; Affinity Biosciences), LC3 (3868; Cell Signaling Technology), MRC1 (ab300621; Abcam), and MCT4 (22787‐1‐AP; Proteintech) were commercially purchased.

Techniques: Methylation, Immunohistochemistry, Two Tailed Test, Expressing, Microarray

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