Journal: Stem Cells and Development

Article Title: Normothermic Machine Perfusion Combined with Bone Marrow Mesenchymal Stem Cells Improves the Oxidative Stress Response and Mitochondrial Function in Rat Donation After Circulatory Death Livers

doi: 10.1089/scd.2019.0301

Figure Lengend Snippet: BMMSCs inhibited the JNK-NF-κB pathway and promoted AMPK activation in IAR20 cell after oxidative stress. (A) Western blot of p-JNK (Thr183/Tyr185), JNK, p-p65 (Ser36), and p65 in IAR20 cells (p-JNK, I group: 0.32 ± 0.14, IH group: 0.45 ± 0.13, IH B group: 0.19 ± 0.08, n = 3; p-p65, I group: 0.66 ± 0.33, IH group: 1.03 ± 0.12, IH B group: 0.53 ± 0.13, n = 3). (B) Western blot of p-AMPK (Thr172), AMPKα, p-ACC (Ser79), and ACC in IAR20 cells (p-AMPK, I group: 0.64 ± 0.12, IH group: 0.32 ± 0.11, IH B group: 0.82 ± 0.16, IH B C group: 0.34 ± 0.23, n = 3; p-ACC, I group: 0.86 ± 0.17, IH group: 0.49 ± 0.31, IH B group: 0.99 ± 0.37, IH B C group: 0.43 ± 0.26, n = 3). $ P < 0.05 versus IH group, & P < 0.05 versus IH B group. ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase;; C, Compound C, I, IAR20; IH, IAR20 (H 2 O 2 ); IH B, IAR20(H 2 O 2 ) cocultured with BMMSCs; IH B C, IH B plus Compound C; JNK, JUN N-terminal kinase.

Article Snippet: The following reagents and kits were used in the present study: Dulbecco's modified Eagle's medium (DMEM)/F12 medium (1:1); 0.25% trypsin–EDTA solution (Gibco, Carlsbad, CA, USA); penicillin–streptomycin solution (HyClone, Logan, UT, USA); fetal bovine serum (FBS; Biowest, Loire Valley, France); adipogenic and osteogenic differentiation medium (Sigma-Aldrich, Merck KGaA, St. Louis, MO, USA); Oil Red O (Beijing Dingguo Changsheng Biotechnology, Beijing, China); a von Kossa cell staining kit (Genmed, Shanghai, China); rat green fluorescent protein genomic adenovirus (GFP-Adv, GeneChem, Shanghai, China); proteinase K and a bicinchoninic acid (BCA) protein assay kit (Beijing Solarbio Science & Technology, Beijing, China); western blotting-related reagents, a reactive oxygen species (ROS) assay kit, a tissue mitochondrial isolation kit, and a mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China); anti-rat CD34-fluorescein isothiocyanate (FITC), anti-rat CD29-phycoerythrin (PE), anti-rat CD45-PE, anti-rat CD90-FITC, anti-rat RT1 class I gene (A-1) (RT1A)-PE, anti-rat RT1 class II, locus B (RT1B)-FITC (BioLegend, San Diego, CA, USA); JUN N-terminal Kinase (JNK) mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phosphorylated (p)-nuclear factor kappa B (NF-κB) (p-p65) rabbit antibody, p-JNK rabbit antibody, p-AMP-activated protein kinase (AMPK) rabbit antibody, p -acetyl-CoA carboxylase (ACC) rabbit antibody, ACC rabbit antibody (Cell Signaling Technology, Boston, MA, USA); NF-κB (p65) rabbit antibody, AMPKα rabbit antibody, myeloperoxidase (MPO) (Proteintech, Wuhan, China); β-actin mouse antibody, goat anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-HRP), goat anti-mouse IgG-HRP, FITC-conjugated goat anti-rabbit IgG, streptavidin–peroxidase kit (ZSGB-BIO, Beijing, China); malondialdehyde (MDA) and glutathione (GSH) assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); a annexin V-FITC/PI apoptosis detection kit (Keygen Biotech, Nanjing, China); inverted fluorescent microscope (Olympus, Tokyo, Japan); Eclipse Ni-U-positive fluorescence microscope (Nikon); BD Accuri C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA); Molecular Imager ChemiDoc XRS+ system (Bio-Rad, Foster City, CA, USA); an in vivo imaging system (PerkinElmer, Fremont, CA, USA); and a transmission electron microscope (Hitachi, Ltd., Tokyo, Japan).

Techniques: Activation Assay, Western Blot

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: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Super‐resolution fluorescence microscopy images showing relative distribution between MATR3 and DAPI. Scale bars, 5 μm (Upper) or 0.5 μm (Lower). Arrows point to the representative MATR3/DAPI co‐staining regions. B Coefficient of correlation between MATR3 and histone modification H3K9me3 ( n = 105), H3K9me2 ( n = 98), H3K27me3 ( n = 107), H3K27ac ( n = 97) and H3K4me3 ( n = 99) in AML12 cells. Quantifications were performed on randomly selected ROIs in cell nuclei. Also see Fig . Each point represents one cell. C (Upper) Schematic diagram of dox‐inducible shRNA system for MATR3 knockdown and MATR3 rescue in AML12 cells. (Lower) Western blotting detected the expression level of MATR3 after 3 days of Dox treatment (+Dox) and followed by 3 days of Dox removal (±Dox) in AML12 cells. Rep, replicate. D (Upper) Representative cross‐section images showing the distribution of histone modifications upon Ctrl and MATR3 knockdown (+Dox). (Lower) Quantify the distribution pattern of histone modifications by Standard Deviation of Pixel Intensity in cell nuclei. For H3K9me3, n = 102 (Ctrl) or 84 (+Dox); for H3K9me2, n = 100 (Ctrl) or 101 (+Dox); for H3K27me3, n = 98 (Ctrl) or 98 (+Dox); for H3K27ac, n = 117 (Ctrl) or 124 (+Dox); for H3K27me3, n = 98 (Ctrl) or 98 (+Dox); for H3K4me3, n = 107 (Ctrl) or 97 (+Dox). Each point represents one cell. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, not significant, * P < 0.05, **** P < 0.0001. Error bars indicate mean ± s.e.m. E Representative cross‐section images showing nuclear localization of MATR3 and H3K27me3 upon Ctrl, MATR3 knockdown (+Dox) and MATR3 rescue (±Dox). Scale bars, 5 μm. F Relative distribution of H3K27me3 and Lamin A/C. L 0 , region between nuclear membrane (position of nuclear membrane was determined by the X‐axis of the Lamin A/C pixel peaks on both sides). L 1 , region between two H3K27me3 pixel peaks that closest to the nuclear membrane. Quantify changes in H3K27me3 distribution adjacent to nuclear peripheral in Ctrl ( n = 95), MATR3 knockdown (+Dox) ( n = 92) and MATR3 rescue (±Dox) ( n = 98) cells by formula of (L 0 ‐L 1 )/2L 0 (%). Scale bars, 5 μm. Each point represents one cell. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, not significant, **** P < 0.0001. Error bars indicate mean ± s.e.m. G Relative distribution of H3K27me3 and C23. N 0 , region between nucleolus membrane (position of nucleolus membrane was determined by the X‐axis of the half‐peaks on both sides). N 1 , region between two H3K27me3 pixel peaks that are closest to the nucleolus membrane. Quantify changes in H3K27me3 distribution adjacent to nucleolus in Ctrl ( n = 91), MATR3 knockdown ( n = 91) and MATR3 rescue ( n = 92) cells by formula of (N 1 ‐N 0 )/2N 0 (%). Scale bars, 5 μm. Each point represents one cell. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, not significant, **** P < 0.0001. Error bars indicate mean ± s.e.m. H H3K27me3 modification level upon Ctrl and MATR3 knockdown (+Dox) as detected by western blotting. Rep, replication. I Average enrichment of H3K27me3 ChIP‐seq signal at peaks regions with 5 kb upstream and downstream flanking regions in Ctrl and shMatr3 from AML12 cells. J (Upper) Schematic diagram of IAA‐inducible rapid protein degradation system for MATR3 in ES cells. (Lower) Western blotting detection of the efficiency of MATR3 knockdown in ES cells after 6 h addiction of 500 μM IAA or equal volume of alcohol (−IAA). Rep, replication. K Representative cross‐section images showing nuclear localization of H3K27me3 in ES cells after 6 h addiction of 500 μM IAA (+IAA 6 h) or equal volume of alcohol (−IAA). Middle: The zoom‐in view of H3K27me3 in one ES cell. The outer dotted line colocalizes with nuclear membrane; the inner dotted line has the 80% diameter of the outer dotted line. The average pixel intensity of regions within outer dotted line (ROI.o) and regions within inner dotted line (ROI.i) were measured separately for each cell. Scale bars, 5 μm. L Statistics for relative intensity (ROI.o/ ROI.i) after 6 h addiction of 500 μM IAA (+IAA 6 h) ( n = 46) or equal volume of alcohol (−IAA) ( n = 38). Each point represents one cell. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; **** P < 0.0001. Error bars indicate mean ± s.e.m. M Standard deviation of H3K27me3 pixel intensity after 6 h addiction of 500 μM IAA (+IAA 6 h) ( n = 68) or equal volume of alcohol (−IAA) ( n = 60). Each point represents one cell. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; **** P < 0.0001. Error bars indicate mean ± s.e.m. Source data are available online for this figure.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Fluorescence, Microscopy, Staining, Modification, shRNA, Knockdown, Western Blot, Expressing, Standard Deviation, Two Tailed Test, Membrane, ChIP-sequencing

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Immuno‐electron microscopy analysis of MATR3 protein distribution labeled by DAB in the AML12 cell. The solid arrows point to the individual DAB signal and the hallow arrows point to the clustered DAB signals, both of which represent MATR3 proteins. NM, nuclear membrane; Nu, nucleolus. Scale bars, (i): 2 μm; (ii): 0.5 μm; (iii), (iv): 0.2 μm. B (Upper) Super‐resolution fluorescence microscopy images showing relative distribution between MATR3 and histone modifications (H3K9me3, H3K9me2, H3K27me3, H3K27ac and H3K4me3) in AML12 cells. (Lower) Line charts showing pixel intensity of each channel on the regions of interest (ROI). r , coefficient of correlation. Scale bars, 5 μm. C (Left) Representative cross‐section images showing nuclear localization of MATR3 and H3K27me3 upon Ctrl, MATR3 knockdown (+Dox) and MATR3 rescue (±Dox). (Right) Line charts showing pixel intensity of each channel on the ROIs. Scale bars, 10 μm. D Pixel intensity of ROIs showing relative distribution of H3K27me3 and Lamin A/C upon Ctrl, MATR3 knockdown (+Dox) and MATR3 rescue (±Dox). L 0 , region between nuclear membrane. L 1 , region between two H3K27me3 pixel peaks that closest to the nuclear membrane. Scale bars, 5 μm. E Pixel intensity of ROIs showing relative distribution of H3K27me3 and C23. N 0 , region between nucleolus membrane (position of nucleolus membrane on X‐axis determined by C23 pixel half‐peaks on both sides). N 1 , region between two H3K27me3 pixel peaks that closest to the nucleolus membrane. Scale bars, 5 μm. F Standard deviation of H3K27me3 pixel intensity of Ctrl ( n = 98), MATR3 knockdown (+Dox) ( n = 100) and MATR3 rescue (±Dox) ( n = 94). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; * P < 0.05, **** P < 0.0001. Error bars indicate mean ± s.e.m. G Genome browser of the H3K27me3 enriched region in Ctrl and MATR3 knockdown (+Dox) samples. H Immunofluorescent detection of the efficiency of MATR3 knockdown in ES cells after 6 h addiction of 500 μM IAA or equal volume of alcohol (−IAA). Scale bars, 10 μm.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Immuno-Electron Microscopy, Labeling, Membrane, Fluorescence, Microscopy, Knockdown, Standard Deviation, Two Tailed Test

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Western blotting showing the distribution of MATR3 proteins in chromatin‐non‐associated and chromatin‐associated extracts before and after DRB (75 μM for 12 h) or RNase A (pretreat with 0.05% Triton x‐100 for 30s, followed by 10 μg/ml RNase A for 1 h) treatment in AML12 cells. Representative of two independent replicates with similar results. B (Left) The representative cross‐section image showing nuclear distribution of DAPI, MATR3 and H3K27me3 before and after 24 h treating of 75 μM DRB in AML12 cells. (Right) Line charts showing pixel intensity of each channel on the ROIs. r , coefficient of correlation. Scale bars, 5 μm. C Pie chart showing the MATR3 RIP‐seq peaks in relation to the genomic features. For each MATR3 RIP‐seq peak, we found its overlap with the genomic features and annotated. The priority of features is TSS, UTR, exonic, intronic, repeats, intergenic and others. D The number of MATR3 RIP‐seq peaks in repetitive elements (REs) from AML12 cells. E Heatmap of MATR3 RIP‐seq sense and antisense signal of repetitive elements from AML12 cells. All RE copies with the RIP (MATR3 ‐IgG) count number ≥ 10 are kept. For each RE family, copies from antisense and sense of two replicates are merged. Then, compute the RIP (MATR3 ‐IgG) count number for RE copies and compute the mean count of RIP (MATR3 ‐IgG) of each sample. The color indicates the mean reads count of RIP (MATR3 ‐IgG) for each RE family. F Heatmap of MATR3 RIP‐seq antisense and sense signal for MATR3 associated L1 subfamilies. L1 subfamilies are considered as MATR3 associated if the subfamily contains more than 50 copies. The copies with RIP (MATR3 ‐IgG) count number ≥ 10 are kept. L1 RNAs are ranked by antisense mean reads count. The color indicates the mean reads count of RIP (MATR3 ‐IgG) for each L1 subfamily. G (Left) Representative cross‐section images showing relative distribution between AS L1 RNA with MATR3 and with histone modification marks (H3K27me3, H3K9me3 and H3K27ac) in AML12 cells. Probes for RNA FISH were designed toward the consensus sequence of antisense L1_Mus1 RNAs. (Right) Line charts showing pixel intensity of each channel on the ROIs. Scale bars, 5 μm. r , coefficient of correlation. H Normalized average density of the marks (top) and heatmaps(bottom) for the two groups of L1 loci. The two groups are antisense L1 RNAs or sense L1 RNAs that interacted with MATR3. L1 loci with RIP (MATR3 ‐IgG) count number ≥ 10 of antisense RNA were identified as MATR3‐associated antisense L1, and the same cutoff for MATR3‐associated sense L1. Source data are available online for this figure.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Western Blot, Modification, Sequencing

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Representative cross‐section images showing distribution pattern for MATR3 in cell nuclei after 0, 1, 2, 6, 12, 24 h treating with 75 μM DRB. Scale bars, 10 μm. B Statistics for MATR3 distribution pattern in cell nuclei after 0 ( n = 500), 1 ( n = 578), 2 ( n = 560), 6 ( n = 495), 12 ( n = 489), 24 ( n = 490) hours treating with 75 μM DRB. C Heatmap of MATR3 RIP‐seq sense and antisense mean reads count in repetitive elements in ES cells. All RE copies with the RIP (MATR3 ‐IgG) count number ≥ 10 are kept. For each RE family, RE copies from antisense and sense of two replicates are merged. Then, compute the RIP (MATR3 ‐IgG) count number for RE copies and compute the mean count of RIP (MATR3 ‐IgG) of each sample. D Genomic distribution of MATR3‐AS L1 RNAs loci in AML12 cells. E ( Left) Genome browser of the MATR3 RIP, RepeatMasker and Refseq genes. Targeting loci of the primers were noted on the screenshot. (Right) DNA gel images of RT–PCR of MATR3 RIP products (and input RNAs). F Scatter plot of RIP‐seq antisense for MATR3‐associated L1 copies (AS L1) and host genes' expression. ASL1 copies that with RIP (MATR3 ‐IgG) count number ≥ 10 and located within gene body regions are kept. X ‐axis: RIP (MATR3 ‐IgG) count of ASL1 copies. Y ‐axis: the FPKM of ASL1's host genes as detected by RNA‐seq. Pearson correlation coefficient (PCC) between RIP (MATR3 ‐IgG) count number of ASL1 copies and FPKM of host genes. G RNA decay curve for AS L1 RNAs, Neat1 RNA and Actb RNA after blocking transcription with 5 μg/ml actinomycin D before and after MATR3 depletion. RNA remaining was measured relative to 18 s RNA at 0 h by qPCR. The RNA decay assay was performed in three independent biological replicates ( N = 3). Fit modeled by one‐phase decay using nonlinear least squares regression. Error bars indicate mean ± s.d. H (Left) Representative cross‐section images showing relative distribution between AS L1 RNA with MATR3 in ES cells. (Right) Line charts showing pixel intensity of each channel on the ROIs. Scale bars, 5 μm.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing, Blocking Assay

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Representative RNA FISH (with probes targeting antisense L1_Mus1 RNAs) and immunofluorescence images showing nuclear distribution of AS L1 RNAs and MATR3 after 24 h treating with different ASOs in AML12 cells. Scales bars, 10 μm. B Statistical data for intracellular distribution of MATR3 proteins after 24 h treating with Scramble ( n = 200), antisense L1_Mus1 ( n = 198), sense L1_Mus1 ( n = 195), antisense L1_MA7 ( n = 200) and sense L1_MA7 ( n = 200) in AML12 cells. C The representative intracellular distribution of MATR3 proteins before and after treating with AS L1 ASOs in AML12 cells. Scales bars, 5 μm. D Statistical data for intracellular distribution of MATR3 proteins after 0 h ( n = 1,000), 6 h ( n = 1,280), 12 h ( n = 1,504) and 24 h ( n = 1,430) treating with AS L1 ASOs in AML12 cells. E, F The representative cross‐section image showing nuclear distribution of MATR3 and HNRNPU (E) and SAFB (F) before and after 24 h treating with antisense L1 ASOs in AML12 cells. Scales bars, 5 μm. G (Left) The representative cross‐section image showing nuclear distribution of MATR3 and FUS before and after 24 h treating with antisense L1 ASOs in AML12 cells. (Right) The zoom‐in MERGE image showing relative distribution of MATR3 and FUS. Line charts showing pixel intensity of each channel on the ROIs. Scales bars, 5 μm. H Scatter plot of antisense RNA expression changes in L1_Mus1 subfamily with genes expression of host between AS L1 ASO and scramble ASO treatment samples. Mus1 copies located within gene body regions are kept. X ‐axis: antisense RNA expression fold changes log 2 (AS L1 ASO/scramble ASO) of Mus1 copies. Y ‐axis: the FPKM fold changes in L1_Mus1's host genes. Pearson correlation coefficient (PCC) between antisense RNA expression changes in Mus1 copies and expression changes in host genes.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Immunofluorescence, RNA Expression, Expressing

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A (Left) The representative cross‐section image showing nuclear distribution of MATR3 and H3K27me3 before and after 12 h treating with antisense L1 ASOs in AML12 cells. (Right) Line charts showing pixel intensity of each channel on the ROIs. r, coefficient of correlation. Scale bars, 5 μm. B Standard deviation of H3K27me3 pixel intensity before ( n = 98) and after ( n = 97) 12 h treating with antisense L1 ASOs in AML12 cells. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; **** P < 0.0001. Error bars indicate mean ± s.e.m. C (Left) The representative cross‐section image showing nuclear distribution of H3K27me3 and AS L1 RNA before and after MATR3 knockdown (Dox treatment for 3d) in AML12 cells. (Right) The normal distribution curve for the AS L1 pixel intensity. Scale bars, 5 μm. D Skewness of antisense L1 RNA pixel intensity in Ctrl ( n = 63) and shMatr3 cells ( n = 66). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, **** P < 0.0001. Error bars indicate mean ± s.e.m. E Representative images showing nuclear colocalization of AS L1 RNAs with wild‐type and truncated GFP‐MATR3 proteins in AML12 cells. Scale bars, 5 μm. F Coefficient of correlation between AS L1 RNA with wild‐type and truncated GFP‐MATR3 proteins. WT ( n = 98), ▵ZF1 ( n = 83), ▵ZF2 ( n = 89), ▵RRM1 ( n = 91), ▵RRM2 ( n = 94). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; * P < 0.05, **** P < 0.0001. Error bars indicate mean ± s.e.m. G Representative cross‐section images showing nuclear localization of H3K27me3 upon Ctrl, MATR3 knockdown and exogenous GFP‐MATR3 (WT/▵ZF1/▵ZF2/▵RRM1/▵RRM2) overexpression in AML12 cells. Scale bars, 5 μm. H Standard deviation of H3K27me3 pixel intensity upon Ctrl ( n = 50), MATR3 knockdown ( n = 50), GFP‐MATR3 (WT) rescue ( n = 48), GFP‐MATR3 (▵ZF1) rescue ( n = 47), GFP‐MATR3 (▵ZF2) rescue ( n = 46), GFP‐MATR3 (▵RRM1) ( n = 45) and GFP‐MATR3 (▵RRM2) rescue ( n = 49). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, not significant, ** P < 0.01, **** P < 0.0001. Error bars indicate mean ± s.e.m. Source data are available online for this figure.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Standard Deviation, Two Tailed Test, Knockdown, Over Expression

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Representative images of the GFP‐MATR3 (WT/▵RRM2) FRAP experiments in AML2 cells. Solid arrows indicate the bleached points. Scale bars, 5 μm. B The fluorescence recovery curve of the GFP‐MATR3 (WT/▵RRM2) corrected for fluorescence decay. Data are expressed as the mean ± s.e.m. (WT, n = 11; ▵RRM2, n = 11). C Representative images of droplet formation assays with different concentrations of GFP‐MATR3 proteins. NaCl concentration, 50 mM. Scale bars, 5 μm. D Representative images of droplet formation assays with different NaCl concentrations. GFP‐MATR3 protein concentration, 3 μM. Scale bars, 5 μm. E Areas of MATR3 protein droplets formed in different protein concentration (3 μM: n = 315; 2 μM: n = 196; 1 μM: n = 165; 0.5 μM: n = 50). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; **** P < 0.0001. F Areas of MATR3 protein droplets formed in different NaCl concentration (50 mM: n = 248; 100 mM: n = 196; 400 mM: n = 127; 800 mM: none). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; **** P < 0.0001. G (Left) Representative images of droplet formation assays by GFP‐MATR3 with different in vitro ‐transcribed RNAs (Sense L1, Antisense L1, Sense B1, Antisense B1, MajSAT). RNA concentration, 200 nM. GFP‐MATR3 protein concentration, 3 μM. NaCl concentration, 50 mM. (Right) The table showing the nucleotide number and the number of MATR3‐binding motifs on the in vitro ‐transcribed RNAs. Scale bars, 5 μm. H Representative images of droplet formation assays by GFP‐MATR3 with different concentration (0, 5, 50, 200, 500 nM) of AS L1 RNAs. GFP‐MATR3 protein concentration, 3 μM. NaCl concentration, 50 mM. Scale bars, 5 μm. I Schematic representation for MATR3‐MATR3 droplets formation and MATR3‐AS L1 RNA meshwork formation. Source data are available online for this figure.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Fluorescence, Concentration Assay, Protein Concentration, Two Tailed Test, In Vitro, Binding Assay

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Percentages of compartment status at unswitched A, unswitched B, B switched to A and A switched to B between Ctrl and shMatr3. B Average contact enrichment between pairs of 100 kb loci in Ctrl, shMatr3 and the comparison between them. All the 100 kb loci are arranged by Ctrl PC1 values in decreasing order and divided into 50 quantiles. Average enrichment of PC1 values is calculated for each quantile. C Changes in contacts between compartment regions from the same (AA or BB) and different (AB) type in Ctrl and shMatr3. Data are represented as boxplots based on log 2 (O/E‐shMatr3/O/E‐Ctrl) values per pair. The number of compartment A and B are 1,456 and 1,573. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. D Changes in contacts within A or B compartment regions between Ctrl and shMatr3. Data are represented as boxplots based on log 2 (O/E‐shMatr3/O/E‐Ctrl) values per compartment region. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. E Snapshot of an example region, showing Hi‐C, H3K27me3 ChIP‐seq and AS L1 in MATR3 RIP‐seq in Ctrl and/or shMatr3 samples, using HiCExplorer. The values on the y‐axis for Hi‐C contact and O/E heatmap are iced normalized read counts at 100 kb resolution. The values on the y‐axis for ChIP‐seq and RIP‐seq are average reads per million of mapped reads (RPM). F Venn diagram shows the common and sample‐specific TADs between Ctrl and shMatr3 samples. The TADs that overlapped length/TAD length > 0.8 both in Ctrl and shMatr3 samples were identified as common TADs. G Insulation strength at TAD boundaries within compartment A, B regions or AB boundaries. H Heatmap showing six groups of TADs based on average enrichment of AS L1 MATR3 RIP‐seq signal and H3K27me3 ChIP‐seq signal from ctrl cells. Enrichment of AS L1 in MATR3 RIP‐seq classified into strong enriched (++), enriched (+) and none (−) levels. Enrichment of H3K27me3 classified into enriched (+) and none (−) levels. Average enrichment of intra‐TAD contact and PC1 is calculated for each TAD. I Intra‐TAD contacts changes in TADs associated with MATR3‐AS L1 RNAs TADs and nonassociated TADs according to density of anti‐MATR3 RIP‐seq signal of AS L1 RNAs. The y ‐axis showing log2 contacts changes between shMatr3 and ctrl cells. The contacts are the density of the observed contacts normalized by the density of the expected contacts. The number of TADs associated with MATR3‐AS L1 RNAs and nonassociated are 1,658 and 1,486. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. J Intra‐TAD contacts changes in TADs nonassociated with MATR3‐AS L1 RNAs and four quantile groups associated with MATR3‐AS L1 RNAs ranking by increasing in MATR3‐AS L1 RNAs density. The number of TADs nonassociated with MATR3‐AS L1 RNAs and associated four quantile groups is 1,486, 415, 415, 415, and 413. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. K Percentage of TADs located in compartment A or B regions from five TAD groups (same groups in [J]). L Boxplot shows gene expression changes for TAD groups from (J). The number of genes in each group are 2,050, 2,094, 2,356, 2,071 and 1,605. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. Source data are available online for this figure.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Comparison, Hi-C, ChIP-sequencing, Insulation, Gene Expression

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Venn diagram shows the common and sample‐specific TADs between Ctrl and MATR3‐AID samples from ES cells. The TADs that overlapped length/TAD length > 0.8 both in Ctrl and MATR3‐AID samples were identified as common TADs. B Intra‐TAD contacts changes in MATR3‐AS L1 RNAs associated and nonassociated TADs from ESC cells. For each TAD, we calculate the density of the anti‐MATR3 RIP‐seq signal of AS L1 RNAs. TADs are grouped into “No AS L1” and “with AS L1” by density greater than 0 or not. The number of TADs associated with MATR3‐AS L1 RNAs and nonassociated are 2001 and 639. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. C Intra‐TAD contacts changes in TADs nonassociated with MATR3‐AS L1 RNAs and four quantile groups associated with MATR3‐AS L1 RNAs ranking by increasing in MATR3‐AS L1 RNAs density from ES cells. The number of TADs nonassociated with MATR3‐AS L1 RNAs and associated four quantile groups is 639, 500, 500, 500 and 501. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. D Percentage of TADs located in compartments A or B regions from five TAD groups (same groups in C) from ES cells. E Insulation strength at TAD boundaries within compartment A, B regions or at AB boundaries of Ctrl and MATR3‐AID from ES cells. F Percentages of compartment status at unswitched A, unswitched B, B switched to A and A switched to B between Ctrl and MATR3‐AID from ES cells. G Changes in contacts between compartment regions from the same (AA or BB) and different (AB) type in Ctrl and MATR3‐AID from ES cells. The y ‐axis showing log2 contacts changes between MATR3‐AID and ctrl cells. The contacts are the density of the observed contacts normalized by the density of the expected contacts. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero. H Changes in contacts within A or B compartment regions between Ctrl and MATR3‐AID from ES cells. Data are represented as boxplots based on log 2 (O/E‐MATR3‐AID / O/E‐Ctrl) values per compartment region. The number of compartment A and B are 938 and 991. The box of the boxplot covers the 50% of the values between 1 st quartile and 3 rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represents the median value of each group; Red dashed lines indicate the zero.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Insulation

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Representative Western blot on chromatin fractionation experiments of Ctrl and shMatr3 cells. Each fraction was loaded with the same number of cells. GAPDH, histone H3, Lamin A/C were used as positive controls for S1, S2 and S3, S4, respectively. B Genome browser view of the SAMMY‐Seq and H3K27me3 ChIP‐seq in Ctrl and shMatr3 (replicate 1 and 2 merged) cells at a representative region. Gray boxes under each SAMMY‐seq track showing the SAMMY‐seq domains. The shadows showing the shMatr3 loss SAMMY‐seq domains. C The genome coverage of SAMMY domains (S4. vs. S2) from AML12 cells. SAMMY domains are grouped into common, shMatr3‐lost and shMatr3‐gained domains by their genomic location between ctrl and shMatr3. D, E Average SAMMY‐seq signal (S4 vs. S2) (D) and average H3K27me3 ChIP‐seq signal (E) around SAMMY domains (groups as in C) with 200 kb upstream and downstream flanking regions of ctrl and shMatr3 from AML12 cells. F, G Average H3K27me3 ChIP‐seq signal (S4. vs. S2) (F) and average SAMMY‐seq signal (G) around H3K27me3 domains with 200 kb upstream and downstream flanking regions of ctrl and shMatr3 from AML12 cells. We calculate the overlapped region length between the H3K27me3 domains and three groups of SAMMY domains (same groups in [C]). For each H3K27me3 domain, the ratio of overlap regions (overlap length/H3K27me3 peak length) ≥ 0.6 was assigned to its overlapped SAMMY domains. H Average MATR3 RIP‐seq sense and antisense signal at L1 loci around H3K27me3 domains (groups as in [F]) with 200 kb upstream and downstream flanking regions from AML12 cells. I DEGs and the proportion of DEGs in relative to all genes in common, shMatr3‐lost and shMatr3‐gained SAMMY domains, respectively. J Histogram plots showing the DEGs number in 100 kb bins around SAMMY domains boundaries. Y‐axis is the DEGs number in each bin. Source data are available online for this figure.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Western Blot, Fractionation, ChIP-sequencing

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A (Left) Representative cross‐section images showing relative distribution between MATR3 and PcG proteins BMI‐1 and EZH2. (Right) Line charts showing pixel intensity of each channel on the ROIs. r represents coefficient of correlation. Scale bar, 5 μm. B (Upper) Western blotting of anti‐MATR3 immunoprecipitation (anti‐IgG IP as negative control) products hybridized with MATR3, EZH2 and BMI‐1 antibodies. (Lower) Western blot of anti‐EZH2 immunoprecipitation (anti‐IgG IP as negative control) products hybridized with EZH2 and MATR3 antibodies. C, D The representative cross‐section image showing nuclear distribution of BMI‐1 (C) and EZH2 (D) in Ctrl and shMatr3 (+Dox 3d) cells. Scale bar, 10 μm. E (Upper) Standard deviation of BMI‐1 pixel intensity upon Ctrl ( n = 86) and shMatr3 (+Dox 3d) ( n = 89) cells. **** P < 0.0001, Student's t ‐test. Error bars represent SD. (Lower) Standard deviation of EZH2 pixel intensity upon Ctrl ( n = 86) and shMatr3 (+Dox 3d) ( n = 97) cells. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; **** P < 0.0001. Error bars indicate mean ± s.e.m. F Western blotting showing the distribution of BMI‐1 and EZH2 proteins in chromatin‐non‐associated and chromatin‐associated extracts before and after MATR3 knockdown (+Dox 3d) in AML12 cells. Representative of two independent replicates with similar results. G, H (Left) The representative cross‐section image showing nuclear distribution of BMI‐1 (G) and EZH2 (H), and colocalization with H3K27me3 upon Ctrl and MATR3 knockdown (+Dox 3d). (Right) Line charts showing pixel intensity of each channel on the ROIs. r, coefficient of correlation Scale bar, 5 μm. I The RNA level of Gapdh , Malat1 , Neat1 and Hnf1aos1 in anti‐EZH2 RIP products relative to that in anti‐IgG RIP products as detected by RT–qPCR. For each sample, the relative RNA level was normalized to Actb . The RIP–qPCR assay was performed in six independent biological replicates ( n = 6). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, not significant. Error bars indicate mean ± s.d. J (Upper) Western blotting showing the protein level of MATR3 and BMI‐1 in shCtrl and shBmi‐1 cells; (Lower) Western blotting showing the protein level of MATR3 and EZH2 in shCtrl and shEzh2 cells. K The representative images showing nuclear colocalization of MATR3 with AS L1 RNA in shCtrl, shBMI‐1 and shEZH2 AML12 cells. Scales bar, 5 μm. L Coefficient of correlation between MATR3 and AS L1 RNA in shCtrl ( n = 42), shBMI‐1 ( n = 48) and shEZH2 ( n = 46) AML12 cells. The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, not significant. Error bars indicate mean ± s.e.m.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Western Blot, Immunoprecipitation, Negative Control, Standard Deviation, Two Tailed Test, Knockdown, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: A Schematic diagram of degenerative‐disease‐associated mutations on MATR3 protein. B The order/disorder regions of MATR3 (WT/S85C/F115C) protein predicted by the PONDR algorithm. C MATR3 knockdown and GFP‐tagged WT/S85C/F115C MATR3 protein replacement in N2A cells. The efficiency of endogenous MATR3 knockdown and exogenous GFP‐MATR3 (WT/S85C/F115C) overexpression as detected by western blotting. Representative of two independent replicates with similar results. D Representative images of the GFP‐MATR3 (WT/S85C/F115C) FRAP experiments in N2A cells. Solid arrows point to the bleached points. Scale bars, 5 μm. E The fluorescence recovery curve of the GFP‐MATR3 (WT/S85C/F115C) corrected for fluorescence decay in N2A cells. Data are expressed as the mean ± s.e.m. ( n = 9). F Representative images of droplet formation assays with different concentrations of GFP‐hMATR3 (WT/S85C) proteins. NaCl concentration, 50 mM. Scale bars, 3 μm. G Representative cross‐section images showing nuclear localization of H3K27me3 upon Ctrl, MATR3 knockdown and exogenous MATR3 (WT/S85C/F115C) overexpression in N2A cells. Scale bars, 5 μm. H Standard deviation of H3K27me3 pixel intensity upon Ctrl ( n = 93), MATR3 knockdown ( n = 100), GFP‐MATR3 (WT) rescue ( n = 104), GFP‐MATR3 (S85C) rescue ( n = 82) and GFP‐MATR3 (F115C) rescue ( n = 120). The P ‐values were calculated using unpaired two‐tailed Student's t ‐test; ns, not significant, * P < 0.05, **** P < 0.0001. Error bars indicate mean ± s.e.m. Source data are available online for this figure.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques: Knockdown, Over Expression, Western Blot, Fluorescence, Concentration Assay, Standard Deviation, Two Tailed Test

Journal: EMBO Reports

Article Title: MATR3 ‐antisense LINE1 RNA meshwork scaffolds higher‐order chromatin organization

doi: 10.15252/embr.202357550

Figure Lengend Snippet: This possible model proposes that MATR3 proteins and AS L1 RNAs phase‐separate into a dynamic meshwork, thereby facilitating the 3D organization of chromatin that transcribed AS L1 RNAs.

Article Snippet: As mAID‐tag blocked the antigen‐binding sites of the carboxy‐terminal MATR3 protein, which could not be recognized by Bethyl (A300‐591A), we used the amino‐terminal MATR3 antibody (Abcam, ab51081) as an alternative.

Techniques:

Journal: PLoS ONE

Article Title: High-Efficiency Capture of Drug Resistant-Influenza Virus by Live Imaging of Sialidase Activity

doi: 10.1371/journal.pone.0156400

Figure Lengend Snippet: Zanamivir-resistant mutation (Q136K) or oseltamivir-resistant mutation (H275Y) were introduced in NA gene of zanamivir and oseltamivir-sensitive virus A/USSR/92/1977 (H1N1). The NA genes were transfected to COS7 cells. After 24 hr, the NA-expressed cells were incubated with 100 nM zanamivir or oseltamivir in an SFM at 37°C for 5 min. Then, BTP3-Neu5Ac (20 μM for final concentration) was added to cell culture medium at 37°C for 5 min. Fluorescent images of the cells were observed under UV irradiation by using a fluorescence microscope. A scale bar indicates 200 μm.

Article Snippet: African green monkey kidney COS7 cells (ATCC-CRL-1651, one of the SV40-transfected African green monkey kidney fibroblast-like CV-1 cell lines) were grown in a Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS.

Techniques: Mutagenesis, Virus, Transfection, Incubation, Concentration Assay, Cell Culture, Irradiation, Fluorescence, Microscopy

Journal: Thoracic Cancer

Article Title: Disruption of LPA‐LPAR1 pathway results in lung tumor growth inhibition by downregulating B7‐H3 expression in fibroblasts

doi: 10.1111/1759-7714.15193

Figure Lengend Snippet: LPAR1 and CD276 (B7‐H3) were generally highly expressed in fibroblasts. (a) Clustering analysis of the B7 gene family and LPAR gene family (blue = weaker, red = stronger). In the heat map, the rows represent the 38 fibroblasts from CCLE database (expression 22Q2 Public), and the columns represent the B7 gene family and LPAR gene family. (b) Radar plots demonstrated the differential expression of the LPAR gene family and CD276 in 38 fibroblasts from CCLE. (c) Real‐time PCR data showed the relative mRNA expression of the LPAR gene family in HFL‐1, MRC‐5, LL97A, HLF‐a, A549, H1703, H460 cell lines. (d) Western blot data showed the expression of B7‐H3 and LPAR1 in HFL‐1, MRC‐5, LL97A, HLF‐a, A549, H1703, H460 cell lines. (e) B7‐H3 protein expression was positively correlated with LPAR1 protein expression in HFL‐1, MRC‐5, LL97A, HLF‐a, A549, H1703, H460 cell lines. Data represent mean ± SD. Significance was assessed by two‐sided t ‐test. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, no significance.

Article Snippet: LPAR1 antagonist Ki16425 (Debio 0719) was purchased from MCE.

Techniques: Expressing, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Western Blot

Journal: Thoracic Cancer

Article Title: Disruption of LPA‐LPAR1 pathway results in lung tumor growth inhibition by downregulating B7‐H3 expression in fibroblasts

doi: 10.1111/1759-7714.15193

Figure Lengend Snippet: Lysophosphatidic acid (LPA)‐induced B7‐H3 up‐expression via LPAR1 in fibroblasts. (a) Western blot data showed the expression of B7‐H3 treated with LPA 18:1 (10 μM) for different times in MRC‐5 and HLF‐a cells. (b) Real‐time PCR data showed the validation of siRNAs (50 nM). (c) Western blot data showed the expression of B7‐H3 were transfected with 50 nM siRNAs of LPAR1‐6 in MRC‐5 and HLF‐a cells. (d) Western blot data showed the expression of B7‐H3 treated with LPA 18:1 (10 μM, 24 h) with or without LPAR1 siRNA (50 nM) in MRC‐5 and HLF‐a cells. (e) Western blot data showed the expression of B7‐H3 treated with 10 μM Ki16425 in MRC‐5 and HLF‐a cells. Data represent mean ± SD. Significance compared to negative control was assessed by two‐sided t ‐test. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, no significance.

Article Snippet: LPAR1 antagonist Ki16425 (Debio 0719) was purchased from MCE.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Biomarker Discovery, Transfection, Negative Control

Journal: Thoracic Cancer

Article Title: Disruption of LPA‐LPAR1 pathway results in lung tumor growth inhibition by downregulating B7‐H3 expression in fibroblasts

doi: 10.1111/1759-7714.15193

Figure Lengend Snippet: Silencing of LPAR1 or CD276 (B7‐H3) inhibited lysophosphatidic acid (LPA)‐stimulated fibroblast proliferation. (a) Proliferation data showed that the difference of growth rates of HFL‐1, MRC‐5, LL97A, HLF‐a, A549 and H1703 in 10% fetal bovine serum (FBS) media. (b) Proliferation data showed that after 72 h of MRC‐5 and HLF‐a cells in the presence of LPA 18:1 concentration gradient; MEM media with 10% FBS was a positive control. (c) Proliferation data showed that after 72 h of MRC‐5 and HLF‐a cells treated with LPA 18:1 (10 μM) with or without transfected with 50 nM siRNAs of LPAR1 or B7‐H3. (d) Proliferation data showed that after 72 h of MRC‐5 and HLF‐a cells treated with or without LPA 18:1 (10 μM) and Ki16425 (10 μM). Data represent mean ± SD. Significance compared to negative control was assessed by two‐sided t ‐test. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, no significance.

Article Snippet: LPAR1 antagonist Ki16425 (Debio 0719) was purchased from MCE.

Techniques: Concentration Assay, Positive Control, Transfection, Negative Control

Journal: Thoracic Cancer

Article Title: Disruption of LPA‐LPAR1 pathway results in lung tumor growth inhibition by downregulating B7‐H3 expression in fibroblasts

doi: 10.1111/1759-7714.15193

Figure Lengend Snippet: Knocking out CD276 (B7‐H3) or LPAR1 prevented cancer‐associated fibroblasts (CAFs) from promoting lung cancer cell proliferation in coculture system. (a) Proliferation data showed that after 72 h of A549 and H1703 incubated in conditioned media (CM). The CM was Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) after incubation of MRC‐5 or HLF‐a for 48 h. (b) Schematic diagram of coculture model. (c) Fluorescence images of coculture and monoculture after 72 h. (d) Relative quantitative analysis of fluorescent cells in four random fields of view described in (c). Data represent mean ± SD. Significance compared to negative control was assessed by two‐sided t ‐test. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, no significance.

Article Snippet: LPAR1 antagonist Ki16425 (Debio 0719) was purchased from MCE.

Techniques: Incubation, Modification, Fluorescence, Negative Control

Journal: Thoracic Cancer

Article Title: Disruption of LPA‐LPAR1 pathway results in lung tumor growth inhibition by downregulating B7‐H3 expression in fibroblasts

doi: 10.1111/1759-7714.15193

Figure Lengend Snippet: Knocking out CD276 (B7‐H3) or LPAR1 inhibited cancer‐associated fibroblasts (CAFs) promotion of lung tumor growth in xenograft models. (a) Tumor volumes from A549 cells upon subcutaneous transplantation into NOD‐SCID hosts, with or without fibroblasts in a 1:5 ratio ( n = 5). (b) Mice tumor photograph of (a). (c) Tumor volumes from H1703 cells upon subcutaneous transplantation into NOD‐SCID hosts, with or without fibroblasts in a 1:5 ratio ( n = 5). (d) Overall survival curves of the same mice described in (c). (e) Representative immunohistochemical sections image from the same mice described in (a), left panel for Sirian red staining, middle and right panels stained for FAP and α‐SMA, respectively. Data represent mean ± SD. Stastical significance was assessed by two‐sided t ‐test. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, no significance.

Article Snippet: LPAR1 antagonist Ki16425 (Debio 0719) was purchased from MCE.

Techniques: Transplantation Assay, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: The Trithorax group protein dMLL3/4 instructs the assembly of the zygotic genome at fertilization

doi: 10.1101/242008

Figure Lengend Snippet: A . IDGF4-depleted eggs fail to initiate embryogenesis. Compare with the similar observation recorded in dMLL3/4-depleted eggs (see ). Driver: nos- GAL4. The dashed yellow line delimits the egg’s cytoplasm. Error bars represent standard deviation and asterisks indicate significant difference (unpaired t-test; P < 0.0001). Scale bar: 30 μm. B . IDGF4 is required for normal maternal meiotic completion. Polar body (PB) chromatin morphology was used as read-out for successful meiotic completion. Compare with the similar observation recorded in dMLL3/4-depleted eggs (see ). ANOVA; P < 0.0001. Scale bar: 5 μm. C . IDGF4-depleted eggs assemble mitotically-deficient zygotic chromatin. Such chromatin was arrested at a pre-mitotic state. ANOVA; P < 0.0001. Scale bars: 30 μm (egg) and 5 μm (inset). D . IDGF4 is specifically required for the phosphorylation of histone H3 in zygotic chromatin. Although histone H3 serine 10 phosphorylation (pSer10 H3) was virtually undetectable in zygotic chromatin (Z), the same embryos displayed high levels of this modification in their PB chromosomes. Signal quantification is expressed in fluorescence arbitrary units (AU). Scale bars: 30 μm (egg) and 5 μm (insets). E . IDGF4 depletion does not abrogate the acetylation of histone H4 (H4ac) in zygotic chromatin. For comparison, the early zygotic chromatin of control embryos (mitotic cycle 4-6) is depicted. Scale bars: 5 μm.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-dMLL3/4 (1:1000 dilution, from both the Alexander Mazo and Ali Shilatifard labs) [ , ]; rabbit anti-H3K4me1 (1:5000 dilution, Active Motif 39298); rabbit anti-H3K4me2 (1:5000 dilution, Active Motif 39142); rabbit anti-H3K4me3 (1:500 dilution, Active Motif 39160); rabbit anti-H3 (1:8000 dilution, Cell Signalling Technology #9715) and mouse anti-α-Tubulin (1:50000 dilution, Sigma T6199).

Techniques: Standard Deviation, Modification, Fluorescence

Journal:

Article Title: Generation of Transduction-Competent Retroviral Vectors by Infection with a Single Hybrid Vaccinia Virus

doi: 10.1128/JVI.77.12.7017-7025.2003

Figure Lengend Snippet: (A) Structure of the retroviral vector unit integrated in the triple virus and in virus vrR-XSNegfp. Since the 5′ LTR was modified (the U3 region was replaced with a vaccinia virus early promoter; black arrow), the SacI site usually present in the 5′ LTR was deleted. A SacI site is located in the vaccinia virus genome approximately 6 kb upstream of the integration site of the retroviral vector unit, resulting in the 9.6-kb viral SacI fragment. (B) Structure of the provirus in the transduced cell lines. During transduction, the 5′ LTR is reconstituted in the reverse transcription process, and the SacI site reappears, resulting in the 3.6-kb SacI fragment found in the transduced cell lines. (C) Genomic Southern blots of G418-positive NIH 3T3 clones transduced with cell culture supernatants obtained after infection of different cell types. Clones obtained after transduction with retroviral particles produced in CHO cells (lanes 4 and 5), CV-1 cells (lanes 6 and 7), Vero cells (lanes 8 and 9), and chicken cells (lanes 10 and 11) showed the characteristic 3.6-kb SacI band that indicates an integrated retroviral vector. Lane 1, marker DNA; lane 2, DNA of the virus vrR-XSNegfp harboring only the retroviral vector unit; lane 3, DNA of a nontransduced NIH 3T3 clone. Numbers at the left show the size of the marker bands; numbers at the right show size of the hybridizing bands in the samples (in kilobase pairs).

Article Snippet: The monkey kidney cell lines CV-1 (ATCC CCL-70) and Vero (ATCC CCL-81), the NIH 3T3 cell line (ATCC CRL-1658), Chinese hamster ovary cells (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), human lung cell line MRC5 (CCL-171) and 293 kidney cell line (CRL-1573), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Plasmid Preparation, Modification, Transduction, Clone Assay, Cell Culture, Infection, Produced, Marker

Journal:

Article Title: Generation of Transduction-Competent Retroviral Vectors by Infection with a Single Hybrid Vaccinia Virus

doi: 10.1128/JVI.77.12.7017-7025.2003

Figure Lengend Snippet: Expression of retroviral packaging components by recombinant vaccinia viruses shown by Western blotting. CV-1 cells were infected with the indicated viruses, and total cellular extracts were prepared and subjected to the Western blot procedure. (A) Incubation of the blots with a gag-pol antiserum revealed a characteristic pattern of protein bands, including the Pr65 gag-specific band and the p30 capsid (CA) band (arrowhead, right side) in the packaging virus and two clones of the triple viruses. Lane 1, protein markers. Lane 2, positive control, lysate of cells infected with the virus vHA-MLVg, which has a single insert of the gag-pol sequences (see text). Lane 3, negative control, lysate of cells infected with WR wild-type virus. Lane 4, lysate of cells infected with the packaging virus. Lane 5, lysate of cells infected with triple virus clone 1. Lane 6, lysate of cells infected with triple virus clone 2. (B) Incubation of blots with VSV-G antibodies revealed the typical 55-kDa band of the VSV-G glycoprotein in the packaging and triple viruses. Lane 1, protein markers. Lane 2, positive control, lysate of cells infected with the virus vDD4-mH5-VSVg, a recombinant WR-based virus with a single VSV-G gene insert. Lane 3, negative control, lysate of cells infected with wild-type WR virus. Lane 4, lysate of cells infected with the packaging virus. Lane 5, lysate of cells infected with triple virus clone 1. Lane 6, lysate of cells infected with triple virus clone 2. Numbers at the left indicate the size of the protein markers, and those at the right show the size of characteristic bands (in kilodaltons).

Article Snippet: The monkey kidney cell lines CV-1 (ATCC CCL-70) and Vero (ATCC CCL-81), the NIH 3T3 cell line (ATCC CRL-1658), Chinese hamster ovary cells (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), human lung cell line MRC5 (CCL-171) and 293 kidney cell line (CRL-1573), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Expressing, Recombinant, Western Blot, Infection, Incubation, Clone Assay, Positive Control, Negative Control

Journal:

Article Title: Generation of Transduction-Competent Retroviral Vectors by Infection with a Single Hybrid Vaccinia Virus

doi: 10.1128/JVI.77.12.7017-7025.2003

Figure Lengend Snippet: Colony-forming assays in NIH 3T3 cells. The kidney cell line CV-1 was infected with the indicated viruses, and the vaccinia virus-free supernatants were used to perform colony-forming assays in NIH 3T3 cells with the transduced neomycin resistance marker. Colonies were selected at 500 μg of the antibiotic G418 per ml and stained with crystal violet after 2 weeks. (A) Supernatants of CV-1 cells (the 1:1,000 dilution is shown) infected with the vaccinia virus triple virus gave rise to G418-resistant NIH 3T3 colonies. (B) Supernatants of the same cell line infected with wild-type virus did not result in colonies of the indicator cell line.

Article Snippet: The monkey kidney cell lines CV-1 (ATCC CCL-70) and Vero (ATCC CCL-81), the NIH 3T3 cell line (ATCC CRL-1658), Chinese hamster ovary cells (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), human lung cell line MRC5 (CCL-171) and 293 kidney cell line (CRL-1573), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Infection, Marker, Staining

Journal:

Article Title: Generation of Transduction-Competent Retroviral Vectors by Infection with a Single Hybrid Vaccinia Virus

doi: 10.1128/JVI.77.12.7017-7025.2003

Figure Lengend Snippet: Induction of green fluorescence by infections with triple virus or transduction with retroviral particles induced by triple virus. CV-1 cells directly infected (1 PFU per cell) with the triple virus showed a strong fluorescence 12 h after infection (A), while the wild-type (wt) virus did not induce a signal (B). The characteristic cytopathic effects could be observed in the phase contrast picture (lower panels). Supernatants of CV-1 cells infected with the triple virus were used to transduce NIH 3T3 cells. Green fluorescence of NIH 3T3 clones after 6 days is shown (C). A parallel experiment with the same cells transduced with control supernatants (wild-type virus-infected CV-1 cells) did not result in a signal (D).

Article Snippet: The monkey kidney cell lines CV-1 (ATCC CCL-70) and Vero (ATCC CCL-81), the NIH 3T3 cell line (ATCC CRL-1658), Chinese hamster ovary cells (ATCC CRL-9096), the thymidine kinase-negative cell line 143B (ATCC CRL-8303), human lung cell line MRC5 (CCL-171) and 293 kidney cell line (CRL-1573), and the Western Reserve (WR) strain of vaccinia virus (ATCC VR119) were obtained from the American Type Culture Collection.

Techniques: Fluorescence, Transduction, Infection, Clone Assay

Journal: British Journal of Pharmacology

Article Title: Gα 12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3‐kinase‐mediated activation in a RhoA‐dependent manner

doi: 10.1111/bph.14040

Figure Lengend Snippet: Effects of Gα12 siRNA and p115RhoGEF‐RGS overexpression on M3 receptor‐mediated activation of the PI3K/ROCK/MLC axis in HASMCs. (A–C) Measurement of phosphorylation responses to carbachol (10 μM, 10 min) and protein expression in primary HASMCs after transfection with Gα12 or scrambled siRNA (50 nM, 72 h post‐transfection). (A) Effect of Gα12 or scrambled siRNA on protein expression. Data normalized to tubulin expression in the same samples. (B) Effect of carbachol on Akt, MYPT1 and MLC phosphorylation at S473 (pAkt), T696 (pMYPT1) and S19 (pMLC) after transfection with Gα12 or scrambled siRNA. pAkt, pMYPT1 and pMLC data were normalized to total Akt (Akt), total MYPT1 (MYPT1) and total MLC (MLC). (C) Effect of p115RhoGEF‐RGS overexpression on carbachol‐induced Akt phosphorylation in hTERT‐immortalized HASMCs. Control refers to hTERT‐immortalized HASMCs that underwent G418 selection. (D) Effect of p115RhoGEF‐RGS overexpression on carbachol‐induced intracellular calcium mobilization in hTERT‐immortalized HASMCs. Data are expressed as fold change over untreated (basal) samples that were measured on the same gel or plate. Data (means ± SD) are from five independent donors (n = 5). (E) Effect of p115RhoGEF‐RGS overexpression on carbachol‐induced contraction as measured by MTC analysis in hTERT‐immortalized HASMCs (control, n = 278; p115RhoGEF‐RGS, n = 237). (F) Representative images of a typical modified HASMC responding to carbachol. A single nucleus confirms the presence of one cell. The addition of carbachol induces increased force generation by the cell onto the contractible fluorescent micro‐pattern, resulting in a smaller pattern over time. The white outline in the rightmost figure depicts the initial area of the cell before carbachol stimulation. (G) Quantification of cell contraction to carbachol in p115RhoGEF‐RGS‐expressing and control HASMCs. Line plots depict the evolution of contractile forces, shown as the median population‐wide responsiveness in each of 36 technical experimental replicates (thin grey lines) and their mean (heavy lines) for both the p115RhoGEF‐RGS‐expressing and control HASMCs. Comparison of the heavy lines in the rightmost figure demonstrates a significant inhibition in contractile responsiveness in p115RhoGEF‐RGS‐expressing HASMCs compared with control HASMCs. Bars represent SEM, with each thin grey line representing between 13 and 52 isolated cells analysed per replicate, corresponding to ≥800 total cells analysed per condition. Data (means ± SEM) are from five biological replicates (n = 5). *P < 0.05, significantly different as indicated; in A, D, E unpaired t‐test: in B, C, G, one‐way ANOVA with Bonferroni's post test. Control in all experiments refers to hTERT‐immortalized HASMCs that underwent G418 selection. n.s., not significant; RFU, relative fluorescence unit.

Article Snippet: Antibodies for detection of pMYPT1‐Thr 696 (5163S), pAkt (4060S), pMLC (3674S) and GAPDH (2118S) and total Akt (4691S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, Activation Assay, Expressing, Transfection, Selection, Modification, Inhibition, Isolation, Fluorescence

Journal: British Journal of Pharmacology

Article Title: Gα 12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3‐kinase‐mediated activation in a RhoA‐dependent manner

doi: 10.1111/bph.14040

Figure Lengend Snippet: Effects of M2 receptor siRNA, M3 receptor siRNA and PTX on carbachol‐induced Akt and MLC phosphorylation in primary HASMCs. (A–C) Measurement of phosphorylation responses to carbachol (10 μM, 10 min) and protein expression in primary HASMCs after transfection with M2 receptor, M3 receptor or scrambled siRNA (50 nM, 72 h post‐transfection). (A) Effect of scrambled, M2 receptor and M3 receptor siRNA on protein expression. Data normalized to GAPDH expression in the same samples. (B) Effect of carbachol on Akt phosphorylation at S473 (pAkt) after transfection with scrambled, M2 receptor and M3 receptor siRNA. pAkt data were normalized to total Akt (Akt). (C) Effect of carbachol on MLC phosphorylation at S19 (pMLC) after transfection with scrambled, M2 receptor and M3 receptor siRNA. pMLC data were normalized to total MLC (MLC). (D) Effect of PTX (18 h, 1 μg·mL−1) on carbachol‐induced Akt phosphorylation. Data normalized to tubulin expression in the same samples. (E) Effect of PTX (18 h, 1 μg·mL−1) on carbachol‐induced attenuation of isoprenaline‐mediated cAMP production. Data are expressed as fold change over untreated (basal) samples that were measured on the same gel or plate. Data (means ± SD) are from five independent experiments (n = 5). *P < 0.05, significantly different as indicated; one‐way ANOVA with Bonferroni's post test. n.s., not significant.

Article Snippet: Antibodies for detection of pMYPT1‐Thr 696 (5163S), pAkt (4060S), pMLC (3674S) and GAPDH (2118S) and total Akt (4691S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Transfection

Journal: British Journal of Pharmacology

Article Title: Gα 12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3‐kinase‐mediated activation in a RhoA‐dependent manner

doi: 10.1111/bph.14040

Figure Lengend Snippet: Effects of RhoA inhibitors and siRNA on M3 receptor‐mediated activation of PI3K in primary HASMCs. Measurement of phosphorylation responses to carbachol (10 μM, 10 min) and protein expression in primary HASMCs after transfection with RhoA, Rac1 or scrambled siRNA (50 nM, 72 h post‐transfection) or after incubation with Rhosin (RhoA inhibitor) (10 μM, 30 min). (A) Effect of scrambled, RhoA and Rac1 siRNA on protein expression. Data normalized to MLC expression in the same samples. (B) Effect of carbachol on Akt phosphorylation at S473 (pAkt) after transfection with RhoA, Rac1 or scrambled siRNA. pAkt data were normalized to total Akt (Akt). (C) Effect of Rhosin on carbachol‐induced Akt phosphorylation at S473 (pAkt). Data are expressed as fold change over untreated (basal) samples that were measured on the same gel. Data (means ± SD) are from five independent experiments (n = 5). *P < 0.05, significantly different as indicated; one‐way ANOVA with Bonferroni's post test.

Article Snippet: Antibodies for detection of pMYPT1‐Thr 696 (5163S), pAkt (4060S), pMLC (3674S) and GAPDH (2118S) and total Akt (4691S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Transfection, Incubation