Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Phf6 loss decreases the leukemogenic potential of cells in vivo and triggers a change in disease presentation. ( A ) PHF6 is a lineage-specific regulator of tumor growth in B-ALL and T-cell acute lymphoblastic leukemia (T-ALL). ( B ). Kaplan-Meier survival analysis of mice injected with either 10 3 (dotted) or 10 6 (solid) Phf6 WT (blue) and Phf6 KO (red) B-ALL cells. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in comparison with mice injected with Phf6 WT cells. P- values are shown for the comparisons. ( C ) Representative hematoxylin and eosin (H&E) ( top ) and immunohistochemistry ( bottom ) staining of serial sections from lymph nodes (LNs) and lymphoma (mass) of recipient mice injected with Phf6 WT , shPhf6, and Phf6 KO cells. mCherry immunochemistry demarcates tumor cells. Bars, 600 µm. ( D , top ) Size comparison of representative LNs from Phf6 WT ( left ) and Phf6 KO ( right ) recipient mice. ( Bottom ) Quantification of combined LN weight of Phf6 WT (blue; n = 5) and Phf6 KO (red; n = 5) recipients. ( E ) Tumor burden in the blood of Phf6 WT (blue; n = 7) and Phf6 KO (red; n = 8) recipient mice. mCherry demarcates tumor cells. ( F ) Bar graphs showing the percentage of the CD4 + fraction among mCherry + cells isolated from Phf6 WT (blue; n = 9) and Phf6 KO (red; n = 5) tumors in bone marrow ( left ) and LNs ( right ). Data represent the mean ± standard deviation (SD) in D – F . Statistics were calculated with two-sided Student's t -test. (***) P < 0.001; (****) P < 0.0001.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: In Vivo, Injection, Comparison, Immunohistochemistry, Staining, Isolation, Standard Deviation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: The absence of Phf6 promotes an altered gene expression program in B-cell leukemia. ( A ) Heat map showing differentially expressed genes (fold change >4, false discovery rate [FDR] <0.05) in pairwise comparisons between Phf6 WT ( left ) and Phf6 KO ( right ) cells as determined by RNA-seq. Each column represents a replicate sample. The scale corresponds to row-wise standardized log 2 -transformed expression values for each gene. ( B ) The top gene ontology (GO) and PANTHER terms found to be enriched in Phf6 KO cells. The P -value for each term is plotted as −log 10 ( P -value). ( C ) GSEA plot depicting significant ( P < 0.001) changes in pre-B lymphocyte signature genes upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. ( D ) Quantitative PCR (qPCR) analysis of Phf6 WT (blue) and Phf6 KO (red) cells transduced with empty vector (EV; solid) or a vector expressing Phf6 cDNA (cDNA; dotted). Relative mRNA levels for B-cell-associated genes are shown: Phf6 , Cd22 , Cd74 , Il4ra , Lyn , Ly86 , and Blk . ( E ) Schematic representation of ICA used to identify differential expression signatures (independent components [ICs]) in the integrated RNA-seq data set comprised of Phf6 WT , shPhf6, and Phf6 KO cells. Hinton diagram representation of ICA-derived signatures. Columns denote signatures, and rows denote samples. Colors denote relative directionality of gene expression ([red] up-regulation; [green] down-regulation), and the size of each square represents the magnitude of the contribution of each sample to the respective IC. Each signature is two-sided. Vertical boxes denote statistically significant ( P = 0.01, Mann-Whitney test) independent components. IC2 identified a Phf6 KO -specific gene signature. ( F ) GSEA plot depicting ( P = 0.08) enrichment in T-cell signal transduction signature upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. Data represent the mean ± SD. Statistics for these data were calculated with two-sided Student's t -test. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001; (n.s.) not significant.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Gene Expression, RNA Sequencing, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transduction, Plasmid Preparation, Quantitative Proteomics, Derivative Assay, MANN-WHITNEY

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: PHF6 exerts transcriptional regulation by interacting with histones rather than binding sequence-specific DNA sites. ( A ) Pie chart showing the distribution of 77,749 PHF6-binding sites across genomic regions in B-ALL cells. (TTS) Transcription termination site; (UTR) untranslated region. ( B ) De novo DNA sequence motifs identified in PHF6-bound regions at promoters of differentially expressed genes with their associated P -values. Shown are sequence logos of de novo position-weight matrices found by the MEME motif discovery tool ( left ) or those of known transcription factors whose motifs are found to be most similar to the de novo motif discovery results by Tomtom software ( right ). ( C ) Endogenous coimmunoprecipitation (co-IP) assay of TCF12, NF-κB, and PHF6 in the absence ( top ) or presence ( bottom ) of ethidium bromide (EtBr). Input is 7% of immunoprecipitation lysate. ( D , left ) Metagene tracks of PHF6 ChIP-seq signal averaged over all promoter–TSS tracks grouped by relative expression levels. (Red) High; (green) genomic; (purple) low. ( Right ) Metagene track of H3K27ac ChIP-seq signal averaged over all promoter–TSS regions. Shaded regions around average tracks denote estimates of 95% confidence interval (CI) of the metagene average signals based on resampling. ( E ) Metagene tracks of PHF6 ChIP-seq signal and correlation with histone marks: H3K27ac (blue), H3K4me3 (yellow; GSE66234), H3K27me3 (green). Pearson correlation of PHF6 and histone ChIP-seq signals across 10-kb regions spanning the TSS. Differentially expressed genes (solid line) and genome-wide genes (dotted line) are shown. ( F ) Endogenous co-IP assay of histone H3 and PHF6 in the absence ( top ) or presence ( bottom ) of EtBr. Input is 7% of immunoprecipitation lysate.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Binding Assay, Sequencing, Software, Co-Immunoprecipitation Assay, Immunoprecipitation, ChIP-sequencing, Expressing, Genome Wide

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: PHF6 has distinct binding patterns within lineage-specific genes that result in drastic changes in nucleosome occupancy upon genetic deletion. ( A ) Heat maps comparing gene expression of curated CD19 + B-cell ( left ) and CD4 + T-cell ( right ) gene sets between Phf6 WT ( left ) and Phf6 KO ( right ) cells as determined by RNA-seq. Scale corresponds to row-wise standardized log 2 -transformed expression values for each gene. Each column represents a replicate sample. ( B ) Motif enrichment analysis for DNA regions that undergo significant changes in chromatin accessibility in Phf6 KO cells. ( Top ) DNA motifs with decreased chromatin accessibility upon loss of Phf6 . ( Bottom ) DNA motifs with increased chromatin accessibility upon loss of Phf6 . ( C ) Metagene analysis of nucleosome positions ( top ) and fold enrichment of PHF6 binding ( bottom ) plotted for global analysis ( left ), the CD19 + B-cell gene set ( middle ), and the CD4 + T-cell gene set ( right ) assessed at TSSs ±1-kb genomic regions. ( Top ) Nucleosome positions are shown for Phf6 WT (solid line) and Phf6 KO (dotted line) cells, called by the NucleoATAC algorithm and normalized for batch effects using the chromVAR package. ( Bottom ) Metagene tracks of PHF6 ChIP-seq signal averaged over the TSSs ±1 kb in Phf6 WT cells. (−1/+1) +1/−1 nucleosomes flanking the TSS; (NFR) nucleosome-free region. Gray bars indicate major changes in nucleosome positioning that correspond to enriched PHF6 binding. Shaded regions around average tracks denote estimates of 95% CI of the metagene average signals based on resampling.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Binding Assay, Gene Expression, RNA Sequencing, Transformation Assay, Expressing, ChIP-sequencing

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Chromatin instability allows for aberrant T-cell transcription factor signaling. ( A ) Cell proliferation assay comparing Phf6 WT (blue; up arrow), Phf6 KO (red; up arrow), Phf6 WT + control vector (blue; circle-dotted), Phf6 KO + control vector (red; circle-dotted), Phf6 WT + NICD cDNA (blue; down triangle), and Phf6 KO + NICD cDNA (red; down triangle) cells. n = 3. ( B ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes infected with control vector or activated NICD vector. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in comparison with mice injected with Phf6 WT + control vector cells. P -values are shown for the comparisons. ( C ) Quantification of the combined thymus weight of Phf6 WT + control vector (blue; n = 4), Phf6 KO + control vector (red unfilled; n = 6), and Phf6 KO + NICD cDNA (red patterned; n = 7) recipients. ( D ) Bar graphs showing the percentage of the CD4 + fraction among mCherry + cells isolated from Phf6 WT + control vector (blue; n = 5), Phf6 KO + control vector (red unfilled; n = 4), and Phf6 KO + NICD cDNA vector (red patterned; n = 6) tumors from LNs. Data represent the mean ± SD in C and D . Statistics were calculated with two-sided Student's t -test. (*) P < 0.05; (**) P < 0.01; (****) P < 0.0001.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Proliferation Assay, Control, Plasmid Preparation, Injection, Infection, Comparison, Isolation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Loss of Phf6 results in decreased dependence on the driving oncogene BCR–ABL1 in vivo. ( A ) GSEA plots depicting significant changes in the targets of the BCR–ABL1 fusion signature ( P = 0.0129) and dasatinib resistance signature ( P = 0.0056) upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. ( B ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes with subsequent mock or ponatinib treatment (30 mg/kg daily for four consecutive days). The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P -value is shown for the comparison. ( C ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes with mock or 10 mg/kg doxorubicin (single immunoprecipitation dose) treatment. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P -value is shown for the comparison.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: In Vivo, Injection, Comparison, Immunoprecipitation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Model of PHF6 as a chromatin state regulator, permitting transcription factor binding through chromatin accessibility. ( A ) In wild-type cells, PHF6 binds to the ±1 nucleosome flanking the open TSSs of genes, allowing B-cell-specific transcription factors to bind, drive gene expression, and maintain B-cell identity. Conversely, PHF6 binds nucleosomes surrounding the TSSs of T-cell-specific genes, coordinating chromatin compaction and thus blocking the binding of T-cell-specific transcription factors. ( B ) In the absence of PHF6, chromatin is no longer maintained in an open state, B-cell transcription factors cannot bind, and expression of B-cell identity genes is down-regulated. However, T-cell identity genes are no longer inaccessible, allowing T-cell-specific transcription factors to bind and activate aberrant transcriptional programs.

Article Snippet: A mouse Phf6 cDNA was obtained from OriGene (MC203493) and subcloned into pMSCV-PGK-GFP.

Techniques: Binding Assay, Gene Expression, Blocking Assay, Expressing

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Expression of the indicated eIF2α kinase was reduced in LNCaP cells using gene-specific siRNAs. Two different siRNAs were used for knockdown of each eIF2α kinase and compared to scrambled siRNA control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and is plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were transfected with two different siRNAs targeting GCN2 or a scramble siRNA control and cell lysates were prepared and immunoblotted for the indicated proteins. Molecular weight markers are shown in kilodaltons. The relative levels of p-eIF2α normalized to total eIF2α compared to scramble siRNA control are indicated. ( C ) Expression of GCN2 was knocked-down in LAPC-4, C4-2B, MR49F, 22Rv1, or PC-3 cells using two different siRNAs and compared to scrambled siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) LNCaP cells were treated with indicated concentrations of GCN2iB and cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤0.0001. ( E ) LNCaP cells were treated with GCN2iB (2 µM) or DMSO control for 24 hr and protein lysates were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin as indicated. Relative levels of p-eIF2α normalized to total eIF2α are shown. ( F ) Levels of p-GCN2 were measured in prostate tumor microarrays (Biomax PR1921b and PR807c) using immunohistochemistry (IHC). Staining for p-GCN2-T899 from non-malignant ( N = 33) and malignant PCa tissue ( N = 88) from patients >50 years old was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing p-GCN2-T899 staining of normal and malignant prostate tissues are shown. Scale bars shown are 200 µm (main image) and 20 µm (insert).

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Expressing, Standard Deviation, Transfection, Molecular Weight, Western Blot, Immunohistochemistry, Two Tailed Test, Staining

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were transfected with the indicated gene-specific siRNAs or a scramble control and the protein levels of HRI, PERK, or PKR and p-eIF2α, total eIF2α, and ATF4 were measured by immunoblot. Measurements of actin were used as a protein loading control in the immunoblot experiments. Molecular weight markers are shown in kilodaltons. ( B ) The indicated PCa cell lines were transfected with siRNAs targeting GCN2 or ATF4. Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, or actin as indicated. ATF4 was not detected in the LAPC-4 cells.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Transfection, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Immunoblot analyses for 22Rv1, PC-3, and GCN2 KO clones. Protein lysates were analyzed by immunoblot to measure the levels of GCN2, ATF4, p-eIF2α, total eIF2α, or actin. Molecular weight markers are shown in kilodaltons. The relative levels of p-eIF2α normalized to total eIF2α compared to wild-type (WT) parental control are indicated. ( B ) Growth curve of 22Rv1 WT, 22Rv1 GCN2 KO (clone 7), or 22Rv1 GCN2 KO (clone 7) cells overexpressing GCN2. Data from replicate wells ( N = 5) are shown as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( C ) Protein lysates prepared from the 22Rv1 WT, 22Rv1 GCN2 KO (clone 7), or 22Rv1 GCN2 KO (clone 7) cells expressing GCN2 were analyzed by immunoblot for GCN2, ATF4, ASNS, or actin.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Clone Assay, Molecular Weight, Standard Deviation, Expressing

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Amino acid measurements of LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 8 hr. Bar graphs in the top panel show high abundance amino acids and the lower panel those with lower levels. The heat map on the right shows fold change in amino acid abundance for each biological replicate of GCN2iB-treated LNCaP cells versus the vehicle with the scale showing the highest fold change in yellow and lowest in purple. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), vehicle + essential amino acids (EAA), or GCN2iB (2 µM) + EAA, and cell growth was measured for up to 6 days. Error bars indicate SD ( N = 5). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( C ) Cell cycle analyses of LNCaP cells treated with vehicle, GCN2iB (2 µM), vehicle + EAA, or GCN2iB (2 µM) + EAA for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) Genome-wide tRNA charging analysis (CHARGE-seq) of LNCaP cells treated with vehicle (DMSO), GCN2iB (2 µM), or GCN2iB (2 µM) + EAA for 8 hr. The tRNA charging ratio is shown as a bar graph with fold change compared to vehicle. Only tRNA isoacceptors measured in LNCaP cells are shown. Error bars indicate SD ( N = 4). ( E ) tRNA charging percentage for tRNA His in LNCaP cells treated with vehicle, GCN2iB, or GCN2iB + EAA. Statistical significance was determine using a one-way ANOVA with Tukey’s multiple comparisons ( N = 4); ***p ≤ 0.001, ****p ≤ 0.0001. ( F ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + EAA, or GCN2iB (2 µM) combined with the indicated individual amino acids. Cell growth was measured at 4 days in triplicate wells ( N = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD; ****p ≤ 0.0001. ( G ) Cell cycle analysis of LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + histidine (200 µM), or with media lacking histidine for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( H ) LNCaP cells were cultured in normal media, media supplemented with EAA mix, or media supplemented with histidine (200 µM) for 24 hr. Lysates were analyzed by Immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin. Molecular weight markers are presented in kilodaltons for each immunoblot panel. The relative levels of p-eIF2α normalized to total eIF2α compared to normal media (NM) control are indicated.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Two Tailed Test, Standard Deviation, Genome Wide, Cell Cycle Assay, Cell Culture, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Variant Assay, Molecular Weight, Standard Deviation, Transfection

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Silver stain showing proteins that immunoprecipitated (IP) with FLAG-NUP153. b Co-IP shows FLAG-NUP153 interaction with CTCF, and cohesin subunits, SMC3, SMC1A, and RAD21. NUP153 was pulled down using anti-FLAG antibody. c Schematic showing steps of chromatin fractionation assay in HeLa cells. d NUP153 was detected in the nuclear insoluble fraction (P2) along with CTCF and cohesin. NUP153 detected in the chromatin-associated soluble fraction (S3) following micrococcal nuclease (MNase) treatment of P1 fraction. Ppt, precipitate; Sup, supernatant; Nucleoporin 62, NUP62; loading controls: α-TUBULIN (cytoplasm), Histone H3 (chromatin). Experiments were repeated twice. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Silver Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Fractionation

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Distribution of NUP153 peaks in mouse ES cells. Peaks are categorized as promoters (−2 kb from TSS to +100 bp from TSS), gene body (+100 bp from TSS to +1 kb from transcription termination site (TTS)), intergenic sites (<−2 kb from TSS and >+1 kb from TTS). See Supplementary Data for a list of NUP153 binding sites. b Metagene profiles of mean NUP153 binding at NUP153-positive and NUP153-negative TSS and enhancer (±5 kb), and TAD boundaries (±250 kb) (top). Number and percentage of NUP153 binding sites are presented as a table for the indicated genetic elements (bottom). (See Supplementary Data for NUP153 binding sites at different genetic elements.) c Genome-wide CTCF and SMC3 binding sites were compared in control and NUP153 deficient (KD-1, KD-2) mouse ES cells. d Metagene profiles showing mean CTCF and SMC3 binding across CTCF-positive TSS ( n = 2164), enhancers ( n = 2272), and TAD boundaries ( n = 2238) in control and NUP153 deficient mouse ES cells. e Mean CTCF binding in control and NUP153 KD mouse ES cells were compared and CTCF sites were grouped into two. Group I contained CTCF sites that showed greater mean CTCF binding in control cells over NUP153 KD cells. Group II contained CTCF sites that showed equal or lesser mean CTCF binding in control cells over NUP153 KD cells. Number of CTCF-positive sites across TSS and enhancer (±2.5 kb) and TAD boundaries (±250 kb), and the number of NUP153 target genes that associate with each group are shown as a table (top) (see also Supplementary Data and Supplementary Fig. ). Metagene profiles showing mean NUP153 binding across CTCF-positive Group I and Group II TSS, enhancer (±2.5 kb) and TAD boundaries (±250 kb) (bottom).

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Binding Assay, Genome Wide, Control

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Scatter plot showing expression levels of transcripts in log2[CPM] scale in control and NUP153 KD-1 mouse ES cells. Blue points denote all differentially expressed genes ( n = 711) and orange points denote differentially expressed bivalent genes (Supplementary Data and ). b Table showing number of differentially expressed genes that associate with all, Group I and Group II CTCF-positive TSS (top). Plots showing number of differentially regulated NUP153-positive and NUP153-negative genes that associate with Group I and II CTCF-positive TSS (bottom) (see also Supplementary Data ). c NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq, and RNA-Seq tracks are shown for two NUP153-positive Group I genes, Rtn4rl1 (left panel) and Calb2 (right panel) in control (WT) and NUP153 KD ES cells. Rtn4rl1 shows transcriptional upregulation and Calb2 shown transcriptional downregulation. d NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq tracks are shown for a 145–150 kb region for the HoxA and HoxC loci in control (WT) and NUP153 KD mouse ES cells as indicated. Arrows point to regions where CTCF or SMC3 binding are altered in NUP153 KD mouse ES cells. CTCF sites labeled with asterisk (*) denote CTCF sites that have been reported to regulate transcription at the Hox loci by mediating the formation of TADs , . The 2D heat map shows the interaction frequency in mouse ES cells . Hi-C data was aligned to the mm9 genome showing HoxA cluster residing in a TAD boundary and HoxC cluster in a TAD as published . H3K4me3 and H3K27me3 (ref. ) and CBP/P300 (ref. ) ChIP-Seq data were previously published. CPM, counts per million.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Expressing, Control, ChIP-sequencing, RNA Sequencing, Binding Assay, Labeling, Hi-C

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Western blot showing NUP153 protein levels in control and NUP153 KD HeLa cells. b Real-time RT-PCR showing relative IEG mRNA and nascent mRNA levels in control and NUP153 KD HeLa cells in a time course dependent manner. JUN mRNA levels at minus EGF (control vs KD-1, ** p = 0.0033; control vs KD-2, * p = 0.0345), 15 min EGF (control vs KD-1, * p = 0.0138; control vs KD-2, * p = 0.0199), 30 min EGF (control vs KD-1, *** p = 0.0001; control vs KD-2, *** p = 0.0001), 60 min EGF (control vs KD-1, ** p = 0.0010; control vs KD-2, *** p = 0.0004), 90 min EGF (control vs KD-1, ** p = 0.0065; control vs KD-2, ** p = 0.0045), 120 min EGF (control vs KD-1, ** p = 0.0010; control vs KD-2, ** p = 0.0050). EGR1 mRNA levels at minus EGF (control vs KD-1, ** p = 0.0018; control vs KD-2, *** p = 0.0008), 15 min EGF (control vs KD-1, * p = 0.0373; control vs KD-2, ** p = 0.0066). EGR1 pre-mRNA levels at 15 min EGF (control vs KD-1, ** p = 0.0038; control vs KD-2, * p = 0.0151), 30 min EGF (control vs KD-1, ** p = 0.0045; control vs KD-2, * p = 0.0436), 90 min EGF (control vs KD-1, * p = 0.0364; control vs KD-2, * p = 0.0305). c Real-time RT-PCR showing relative JUN and NUP153 mRNA levels in control, NUP153 KD and FLAG-NUP153 expressing NUP153 KD HeLa cells upon 15 min EGF treatment. GAPDH was used to normalize mRNA levels. JUN mRNA levels in control vs KD (*** p = 0.0000); KD vs FLAG-NUP153 (*** p = 0.0001). NUP153 mRNA levels in control vs KD (** p = 0.0011); KD vs FLAG-NUP153 (*** p = 0.0000). d POL II binding across JUN and EGR1 genetic elements was mapped by ChIP real-time PCR in control and NUP153 KD HeLa cells under indicated conditions (see Supplementary Data for primer sequences). POL II binding at JUN , minus EGF (control vs KD; promoter, * p = 0.0329; TSS, ** p = 0.0042; GB, *** p = 0.0003; TTS, * p = 0.0493), 15 min EGF (control vs KD; promoter, *** p = 0.0009; TSS, ** p = 0.0065; GB, *** p = 0.0009; TTS, * p = 0.0443), 30 min EGF (control vs KD; GB, * p = 0.0385). POL II binding at EGR1 , minus EGF (control vs KD; promoter, * p = 0.0172; TSS, ** p = 0.0060), 15 min EGF (control vs KD; TSS, *** p = 0.0004; TTS, * p = 0.0281). Data shown are percent (%) of input. Values are mean ± standard deviation. Two-tailed Student’s t -test, n ≥ 3 independent experiments. Nt, nucleotide. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: NUP153, CTCF, and SMC3 occupancy across the JUN (left) and EGR1 (right) genetic elements were examined by ChIP real-time PCR at the indicated time points in control and NUP153 KD HeLa cells. Position of PCR primers are denoted as numbers in the schematics (see Supplementary Data for primer sequences). NUP153 binding at JUN , minus EGF (control vs KD; TSS, *** p = 0.0000; GB, ** p = 0.0035; TTS, ** p = 0.0011; +14.6 kb, ** p = 0.0023), 15 min EGF (control vs KD; promoter, ** p = 0.0047; TSS, * p = 0.0368; GB, *** p = 0.0003; TTS, *** p = 0.0004; +14.6 kb, * p = 0.0301). NUP153 binding at EGR1 , minus EGF (control vs KD; −15.3 kb, * p = 0.0298; −1.2 kb, * p = 0.0323; −0.9 kb, ** p = 0.0039; promoter, * p = 0.0469; TTS, * p = 0.0406), 15 min EGF (control vs KD; −15.5 kb, * p = 0.0299; −15.3 kb, ** p = 0.0068; −1.2 kb, *** p = 0.0006; −0.9 kb, ** p = 0.0086; promoter, *** p = 0.0006; TSS, ** p = 0.0078; TTS, ** p = 0.0085). CTCF binding at JUN , minus EGF (control vs KD; GB, ** p = 0.0047; +14.6 kb, * p = 0.0231). CTCF binding at EGR1 , minus EGF (control vs KD; −15.3 kb, ** p = 0.0017; −1.2 kb, ** p = 0.0010; −0.9 kb, * p = 0.0291), 15 min EGF (control vs KD; −15.3 kb, * p = 0.0316). SMC3 binding at JUN , minus EGF (control vs KD; −1.3 kb, ** p = 0.0076; promoter, ** p = 0.0032; TSS, ** p = 0.0037; GB, * p = 0.0413; +14.6 kb, * p = 0.0158), 15 min EGF (control vs KD; −1 kb, * p = 0.0154; TSS, ** p = 0.0029; GB, ** p = 0.0036; +14.6 kb, * p = 0.0245). SMC3 binding at EGR1 , minus EGF (control vs KD; −15.5 kb, *** p = 0.0001; −15.3 kb, * p = 0.0309), 15 min EGF (control vs KD; −15.3 kb, *** p = 0.0004; −1.2 kb, * p = 0.0355; −0.9 kb, * p = 0.0419; TTS, *** p = 0.0003). Data shown are percent (%) of input. Values are mean ± standard deviation. Two-tailed Student’s t -test, n ≥ 3 independent experiments. Nt, nucleotide. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Real-time Polymerase Chain Reaction, Control, Binding Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Real-time RT-PCR showing relative CTCF mRNA levels in HeLa cells transfected with control or CTCF shRNA expression vectors (KD-1 and KD-2). Control vs KD-1, *** p = 0.0000; control vs KD-2, *** p = 0.0000. b Real-time RT-PCR showing relative JUN and EGR1 mRNA levels in control or CTCF KD HeLa cells. JUN mRNA levels at minus EGF (control vs KD-1, * p = 0.0126; control vs KD-2, ** p = 0.0011), 15 min EGF (control vs KD-1, ** p = 0.0077; control vs KD-2, ** p = 0.0011), 30 min EGF (control vs KD-1, *** p = 0.0000; control vs KD-2, *** p = 0.0000), 120 min EGF (control vs KD-2, * p = 0.0299). EGR1 mRNA levels at minus EGF (control vs KD-2, * p = 0.0473), 15 min EGF (control vs KD-1, ** p = 0.0012; control vs KD-2, ** p = 0.0050), 30 min EGF (control vs KD-1, * p = 0.0162; control vs KD-2, *** p = 0.0000). c POL II binding across the IEGs, JUN and EGR1 loci, was mapped by POL II ChIP in control and CTCF KD HeLa cells at the paused state (minus EGF). POL II occupancy at the indicated genetic elements was measured using primers as denoted in the schematics (Supplementary Data ). POL II binding at JUN promoter (control vs KD-1; * p = 0.0261), TSS ((control vs KD-1; ** p = 0.0012), (control vs KD-2; ** p = 0.0087)), GB (control vs KD-2; * p = 0.0463); EGR1 promoter ((control vs KD-1; * p = 0.0209), (control vs KD-2; ** p = 0.0038)), TSS ((control vs KD-1; ** p = 0.0048), (control vs KD-2; ** p = 0.0011)). Data shown are percent (%) of input. d Real-time RT-PCR showing relative EGR1, JUN, NUP153, and CTCF mRNA levels in NUP153 KD, CTCF KD, and in CTCF/NUP153 KD HeLa cells. JUN mRNA levels (control vs NUP153 KD, *** p = 0.0009; control vs CTCF-KD, *** p = 0.0006; control vs NUP153 and CTCF KD, *** p = 0.0002). EGR1 mRNA levels (control vs NUP153 KD, *** p = 0.0003; control vs CTCF-KD, *** p = 0.0000; control vs NUP153 and CTCF KD, *** p = 0.0001). NUP153 mRNA levels (control vs NUP153 KD, *** p = 0.0000; control vs NUP153 and CTCF KD, *** p = 0.0000). CTCF mRNA levels (control vs CTCF-KD, *** p = 0.0000; control vs NUP153 and CTCF KD, *** p = 0.0005). Values are mean ± standard deviation. Relative mRNA levels were normalized using GAPDH. Two-tailed Student’s t -test, n ≥ 3 independent experiments. NS, not significant ( p > 0.05). Nt, nucleotide. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Quantitative RT-PCR, Transfection, Control, shRNA, Expressing, Binding Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding

doi: 10.1038/s41467-020-16394-3

Figure Lengend Snippet: a Immunostaining of LAMIN B1 and c-FOS DNA FISH in control and NUP153 KD HeLa cells are shown at the indicated time points. Cell numbers are as indicated. HeLa cells contain three c-FOS alleles (white arrows). Scale bar, 5 μm. b Cumulative frequency graphs showing distribution of the c-FOS locus distance to nuclear periphery in control and NUP153 KD HeLa cells at the indicated time points. Cumulative frequencies at a normalized distance (ND) of 0.0–0.12 are shown. See Supplementary Fig. for the distribution of loci in all cells that were analyzed (ND of 0.0–0.5). ND = c-FOS locus to periphery distance/cell diameter ( d ), where d = (2× nuclear area/ π ) 0.5 . * p < 0.05; *** p < 0.001; Kolmogorov–Smirnov (KS)-test, n = 2 independent experiments. c Working model showing NUP153-mediated chromatin structure and transcription regulation at the IEG locus. Source data are provided as a file.

Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full-length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP153 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

Techniques: Immunostaining, Control

Journal: bioRxiv

Article Title: Loss of H3K9me3 maintenance in human neural progenitor cells leads to transcriptional activation of L1 retrotransposons

doi: 10.1101/2025.03.28.645885

Figure Lengend Snippet: A Schematic of the CRISPRi construct and workflow for CRISPRi-based silencing of SETDB1 in hNPCs. B qRT-PCR analysis of SETDB1 expression after CRISPRi silencing in hNPCs. Results are shown as means with standard deviation ( n =2 and n =4 respectively). C Western blot analysis of SETDB1 protein levels in SETDB1-CRISPRi and control hNPCs relative to β-actin protein levels. D RPKM normalized genome browser tracks showing: expression of SETDB1 in SETDB1-CRISPRi and control hNPCs (top). Epigenetic changes as a result of the CRISPRi approach as determined by CUT&RUN profiling of H3K4me3 (middle) and H3K9me3 (bottom). E Heat map showing mean expression of NPC, neuronal, and pluripotency gene markers in SETDB1-CRISPRi ( n =2) and control ( n =4) hNPCs as determined by bulk RNA sequencing. F Immunocytochemistry of NESTIN (cyan) and SOX2 (red) in SETDB1-CRISPRi and control hNPCs. Scale bar = 20 µm. G Heat maps illustrating genome-wide RPKM normalized CUT&RUN signal of H3K9me3 and non-targeting control IgG in SETDB1-CRISPRi ( n =2) and control ( n =2) hNPCs. H Heat maps illustrating RPKM normalized CUT&RUN signal of H3K9me3 and non-targeting control IgG in SETDB1-CRISPRi ( n =2) and control ( n =2) hNPCs over full-length L1HS-L1PA3 elements. I Genome browser snapshot of chromosome 8 (top) and chromosome 2 (bottom) showing RPKM normalized CUT&RUN signal of H3K9me3 in SETDB1-CRISPRi and control hNPCs. Right panels show zoom in of H3K9me3 dense areas highlighted in blue. J Immunocytochemistry of H3K9me3 (red) and nuclear marker DAPI (blue) in SETDB1-CRISPRi and control hNPCs. H3K9me3 foci are indicated with white arrows. Scale bar = 10 µm.

Article Snippet: After incubation in blocking solution (KPBS + 0.25% Triton X-100 with 5% normal donkey serum) for 1h at room temperature, the cells were incubated with primary antibodies SOX2 (R&D Systems, AF2018, 1:100) and NESTIN (Abcam, AB176571, 1:100) diluted in blocking solution overnight at 4°C.

Techniques: Construct, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot, Control, RNA Sequencing, Immunocytochemistry, Genome Wide, Marker

Journal: bioRxiv

Article Title: Loss of H3K9me3 maintenance in human neural progenitor cells leads to transcriptional activation of L1 retrotransposons

doi: 10.1101/2025.03.28.645885

Figure Lengend Snippet: A qRT-PCR analysis of SETDB1 expression after CRISPRi g2-based silencing in hNPCs. Results are shown as means with standard deviation ( n =2 and n =4 respectively). B RPKM normalized genome browser tracks showing expression of SETDB1 in SETDB1-CRISPRi g2 and control hNPCs as determined by bulk RNA sequencing C Western blot analysis of SETDB1 protein levels relative to β-actin protein levels in SETDB1-CRISPRi g2 and control hNPCs. D Heat map of NPC, neuronal and pluripotency gene marker mean expression in SETDB1-CRISPRi g2 ( n =2) and control ( n =4) hNPCs as determined by bulk RNA sequencing. E Immunocytochemistry of NESTIN (cyan) and SOX2 (red) in SETDB1-CRISPRi g2 and control hNPCs. Scalebar = 20 µm. F Mean plot showing differential gene expression in SETDB1-CRISPRi ( n =4) compared to control ( n =4) hNPCs. LFC >1, padj < 0.05 calculated with DESeq2. G Gene-ontology overrepresentation test of significantly differentially expressed genes (|LFC| > 1) associated with the selected top upregulated terms (left) and the selected top downregulated terms (right) in SETDB1-CRISPRi compared to control hNPCs. H CUT&RUN analysis of H3K9me3 over genomic regions with well-established H3K9me3-enrichment in SEDTB1-CRISPRi and control hNPCs I Immunocytochemistry of H3K9me3 (red) and nuclear marker DAPI (blue) in SETDB1-CRISPRi g2 and control hNPCs. H3K9me3 foci are indicated with white arrows. Scalebar = 10 µm. J Western blot analysis of H3K9me3 levels relative to Histone 3 in SETDB1-CRISPRi g1 hNPCs compared to control (left) and SETDB1-CRISPRi g2 hNPCs compared to control (right).

Article Snippet: After incubation in blocking solution (KPBS + 0.25% Triton X-100 with 5% normal donkey serum) for 1h at room temperature, the cells were incubated with primary antibodies SOX2 (R&D Systems, AF2018, 1:100) and NESTIN (Abcam, AB176571, 1:100) diluted in blocking solution overnight at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation, Control, RNA Sequencing, Western Blot, Marker, Immunocytochemistry, Gene Expression

Journal: hLife

Article Title: Epigenetic regulation of cardiac tissue development by lysine lactylation

doi: 10.1016/j.hlife.2024.12.005

Figure Lengend Snippet: Figure 5. Genome-wide analysis of the transcriptional consequences of H4K12la in 1 w and 6 w mouse hearts (A) Genome-wide distribution of H4K12la in the mouse hearts at 1 w and 6 w. (B) Curves showing the average profile of H4K12la ChIP-seq read counts around all known TSSs. (C) Heatmap showing differential enrichment of H4K12la ChIP-seq read counts around the TSSs of genes at 6 w compared to those at 1 w. The color scale represents increased enrichment in red and decreased enrichment in blue. The KEGG pathway of genes with differential enrichment of H4K12la ChIP-seq read counts is indicated. (D) Genome browser tracks of the ChIP-seq signal of H4K12la at Mex3b and Vstm5 gene loci. (E) RT-qPCR analysis for target genes in the 1 w and 6 w mouse hearts. Relative expression levels are normalized to Rer1. *P < 0.05, **P < 0.01 (n = 3, unpaired Student’s t-test, values are expressed as the mean ± SD). (F) qPCR analysis of H4K12la ChIP products from the 1 w and 6 w mouse hearts. *P < 0.05, **P < 0.01 (n = 3, unpaired Student’s t-test, values are expressed as the mean ± SD). Abbreviations: ChIP-seq, chromatin immunoprecipitation sequencing; TSS, transcriptional start sites; RT-qPCR, reverse tran- scription quantitative real-time polymerase chain reaction; SD, standard deviation.

Article Snippet: The following antibodies were utilized in this study: anti-l-lactyllysine antibody conjugated agarose beads (PTM Bio, Zhejiang, China; Cat#PTM-1404), anti-l-Lactyllysine rabbit mAb (PTM Bio, Zhejiang, China; Cat#PTM-1401RM), anti-l-lactyl-histone H3 (Lys14) rabbit pAb (PTM Bio, Zhejiang, China; Cat#PTM1414), anti-l-lactyl-histoneH3 (Lys18) rabbitmAb (PTMBio, Zhejiang, China; Cat#PTM-1406RM), anti-l-lactyl-histone H4 (Lys5) rabbit mAb (PTM Bio, Zhejiang, China; Cat#PTM-1407RM), anti-l-lactyl-histone H4 (Lys8) rabbit mAb (PTM Bio, Zhejiang, China; Cat#PTM-1415RM), anti-l-lactyl-histone H4 (Lys12) rabbit mAb (PTM Bio, Zhejiang, China; Cat#PTM-1411RM), antiacetyl-histone H3 (Lys14) mouse mAb (PTM Bio, Zhejiang, China; Cat#PTM-157), anti-acetyl-histone H3 (Lys18) mouse mAb (PTM Bio, Zhejiang, China; Cat#PTM-158), anti-acetyl-histone H4 (Lys5) mouse mAb (PTM Bio, Zhejiang, China; Cat#PTM-163), anti-acetyl-histone H4 (Lys8) mouse mAb (PTM Bio, Zhejiang, China; Cat#PTM-164), anti-acetyl-histone H4 (Lys12) mouse mAb (PTM Bio, Zhejiang, China; Cat#PTM165), anti-histone H3 antibody (Huabio, Zhejiang, China; Cat#M1306-4), E2F2 polyclonal antibody (Invitrogen, California, USA; Cat#PA5-41473), VSTM5 antibody (Biorbyt, Cambridgeshire, UK; Cat#orb313455), RFC3 polyclonal antibody (Invitrogen, California, USA; Cat#PA5-103161), Mex3b antibody (D12) (Santa Cruz, California, USA; Cat#sc-515833), GAPDH (Proteintech, Hubei, China; Cat#60004-1-Ig).

Techniques: Genome Wide, ChIP-sequencing, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: SEMA6A and SEMA6B are host cell receptors for P. sordellii lethal toxin TcsL (A) Genome-wide CRISPR/Cas9 screen in Hap1 cells identifies factors regulating sensitivity to 0.1 nM TcsL. Hap1 cells were infected with a genome-wide TKOv3 gRNA library, treated with recombinant TcsL, and gRNAs from surviving cells were sequenced. (B) Genome-wide CRISPR/Cas9 screen with 1 nM TcsL. (C) Phylogenetic tree of SEMA6 family proteins. (D) Hap1 cells were infected with Cas9 and gRNA targeting indicated genes and tested for sensitivity to TcsL. Data (n = 3) are represented as mean ± standard deviation. Shown at the bottom, expression of SEMA6A and SEMA6B in single and double knockout cell lines was assessed by western blotting. (E) Hap1 SEMA6A KO cells were infected with lentiviruses expressing 3xFLAG-tagged SEMA6 family proteins and tested for TcsL sensitivity. Data (n = 3) are represented as mean ± standard deviation. Shown at the bottom, expression of SEMA6 proteins in infected cell lines was validated with western blotting. See also Figure S1 and .

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Genome Wide, CRISPR, Infection, Recombinant, Standard Deviation, Expressing, Double Knockout, Western Blot

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: Validation of SEMA6A and SEMA6B as host factors required for TcsL intoxication, related to Figure 1 and Figure 2 (A) Expression of SEMA6 family genes, the cognate SEMA6A/6B ligands Plexin A2 and Plexin A4, and known clostridial toxin receptors and host cell factors in Hap1 and HeLa cells based on Human Protein Atlas ( proteinatlas.org ). (B) Left, Sensitivity of SEMA6A and UGP2 knockout cells to TcsL. SEMA6A and UGP2 knockout cells were generated with CRISPR/Cas9. SEMA6A-3xFLAG was ectopically expressed in SEMA6A knockout cells by lentiviral infection. Data (n = 3) are represented as mean ± standard deviation. Right, SEMA6A expression in wild-type Hap1 cells, SEMA6A KO cells, and in SEMA6A KO cells ectopically expressing SEMA6A-3xFLAG. (C) Hap1 and HeLa cells were treated with increasing concentrations of TcsL and cell viability measured 24 h later. Data (n = 3) are represented as mean ± standard deviation. Inset, expression of SEMA6A in Vero, Hap1, and HeLa cells was assessed by western blot. (D) SEMA6A ectodomain protects Vero cells from TcsL toxicity only when added simultaneously with the toxin. SEMA6A and TcsL were added to Vero cells simultaneously or after 1-min or 1 h pre-incubation. Alternatively, TcsL was added for 1 h prior to treatment with SEMA6A. Data (n = 2) are represented as mean ± standard deviation

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Biomarker Discovery, Expressing, Knock-Out, Generated, CRISPR, Infection, Standard Deviation, Western Blot, Incubation

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: SEMA6A and SEMA6B ectodomains protect cells from TcsL intoxication (A) Vero cells stably expressing Nanoluciferase viability reporter were treated with increasing amounts of TcsL in the presence of recombinant SEMA6A ectodomain. Data (n = 2) are represented as mean ± standard deviation. (B) Vero cells stably expressing Nanoluciferase viability reporter were treated with 50 pM TcsL with increasing amounts of recombinant SEMA6 family ectodomains for 24 h. Data (n = 2) are represented as mean ± standard deviation. (C) Microscopy images of Vero cells treated with TcsL (50 pM) and SEMA6 family ectodomains (1 uM) for 24 h. See also Figure S1 .

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Stable Transfection, Expressing, Recombinant, Standard Deviation, Microscopy

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: SEMA6A ectodomain protects lung endothelial cells and mouse lungs from TcsL-induced toxicity (A) SEMA6A and SEMA6B protein expression in human lung endothelial cells. (B) HULECs are extremely sensitive to TcsL. HULEC-5a cells were treated with increased amounts of indicated Clostridial toxins. (C) Recombinant mouse Sema6a ectodomain fused to Fc domain protects HULEC-5a cells from TcsL-induced cell rounding. Cells were treated with 5 pM TcsL and increasing amounts of Sema6a and Sema6c ectodomains. (D) Microscopy images of HULEC-5a cells treated with TcsL and recombinant Sema6a and Sema6c ectodomains. (E) Immunohistochemistry images of Sema6a and Sema6b expression in mouse lung tissue sections. (F) Mice were intraperitoneally injected with 15 ng TcsL and 1,000-fold molar excess of Sema6a ectodomain, Sema6c ectodomain or BSA. Thoracic fluid was collected and measured from symptomatic mice 4 h after injection. (G) Lung tissue sections of mice treated with indicated conditions. Arrows indicate lung edema induced by TcsL.

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Expressing, Recombinant, Microscopy, Immunohistochemistry, Injection

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: Cryo-EM structure of the TcsL 1285–1804 -SEMA6A complex (A) On the left, domain structure of TcsL and SEMA6A. Constructs used in cryo-EM analysis are indicated below. Darker blue color indicates TcsL region resolved to medium and high resolution (< 7 Å) in cryo-EM. Shown on the right is a schematic indicating the location of the TcsL fragment used for cryo-EM, based on full-length TcdB structure (PDB: 6OQ5 ). Abbreviations are as follows: GTD, glucosyltransferase domain; APD, autoprocessing domain; delivery, delivery domain; CROP, combined repetitive oligopeptides; SEMA, semaphorin domain, PSI, plexin-semaphorin-integrin domain; TMD, transmembrane domain. (B) Composite cryo-EM map of the Tcsl-SEMA6A complex. SEMA6A monomers are colored pink and yellow, TcsL domain resolved to medium and high resolution (< 7 Å) is colored dark blue. Low-pass filtered density (10 Å) of the TcsL protein is shown in light blue. Insets I and II show contact residues between SEMA6A (pink) and TcsL (blue). (C) Atomic model built into the SEMA6A-TcsL map. Cryo-EM density of SEMA6A-TcsL is shown as gray mesh with the model built shown as sticks (pink for SEMA6A and blue for TcsL). (D) Comparison of Plexin A2-SEMA6A (PDB: 3OKY ) ( Janssen et al., 2010 ) and TcsL-SEMA6A binding interactions. M109 of SEMA6A interacts with hydrophobic residues of Plexin A2, including L407 and V398 (left). TcsL buries M109 of SEMA6A in a binding pocket containing several hydrophobic residues (middle). An overlay of TcsL and Plexin A2 binding surfaces (shown in blue and purple, respectively) reveals a subset of SEMA6A residues participating in binding to both protein ligands (red). See also Figure S2 , Figure S3 , Figure S4 , Figure S5 .

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Cryo-EM Sample Prep, Construct, Comparison, Binding Assay

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: Biochemical and cryo-EM analysis of the SEMA6A/TcsL complex, related to Figure 4 and Figure 5 (A) Representative binding curves for the TcsL 1285-1804 /SEMA6A interaction. In this analysis, His-tagged TcsL 1285-1804 was immobilized on the Ni-NTA biosensor. The average apparent binding affinity from four independent experiments is 2.4 ± 0.8 nM. The data (blue) were fitted using a 1:1 binding model (red). (B) An example of cryo-EM micrograph. Scale bar is 50 nm. (C) Selected 2D class averages of the TcsL/SEMA6A complex. (D) Gold standard Fourier shell correlation (GSFSC) curve of the final 3D non-uniform refinement of the TcsL/SEMA6A complex in cryoSPARC v2. (E) Viewing direction distribution of the TcsL/SEMA6A data. (F) Selected 2D class averages of SEMA6A dimer. (G) GSFSC curve of the final 3D non-uniform refinement of the SEMA6A dimer in cryoSPARC v2. (H) Viewing direction distribution of the SEMA6A dimer data.

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: Local resolution (Å) plotted on the surface of cryo-EM map of the SEMA6A/TcsL complex and SEMA6A dimer, related to Figure 4 and Figure 5 (A) Local resolution of the SEMA6A/TcsL ranges from 2.8 Å at the core of the SEMA6A to > 30 Å at the flexible terminus of TcsL. (B) Local resolution plotted on the surface of TcsL (left) and SEMA6A (right) binding interfaces. (C) Local resolution plotted on the surface of the SEMA6A cryo-EM map.

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: Comparison of published structures of clostridial toxins and SEMA6A, related to Figure 4 and Figure 5 (A) Left, Cryo-EM structure of TcsL toxin fragment (residues in construct: 1283-1804, residues resolved and modeled: 1400-1637); middle, X-ray structure of TcdB toxin fragment (residues in the model 1283-1804; PDB ID: 6C0B ; Chen et al., 2018 ); right, X-ray structure of TcdA toxin fragment (residues in the model 1-1832; PDB ID: 4R04 ; Chumbler et al., 2016 ). Binding interfaces of TcsL and TcdB to their respective receptors are highlighted in light blue. “H” and “S” letters denote the positions of selected α helices and β strands conserved in all three toxin structures. The glucosyltransferase domain is highlighted in pink, the autoprotease domain is highlighted in green and the delivery domains are highlighted in gray. (B) Comparison of SEMA6A in different structures. All atom RMSD values were calculated using Pymol and are plotted on the surface of SEMA6A dimers (left) and monomers (right).

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Comparison, Cryo-EM Sample Prep, Construct, Binding Assay

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: Clostridial toxins use the same region to bind their cognate receptors (A) Comparison of the TcsL-SEMA6A and TcdB-FZD2 (PDB: 6C0B ) ( Chen et al., 2018 ) binding interface. Residues mutated in TcsL 4mut are indicated in red. (B) TcsL buries M109 of SEMA6A in a hydrophobic binding pocket (left), whereas TcdB utilizes a similar hydrophobic binding pocket to interact with the palmitoleic acid moiety of FZD2 (right). (C) Experimental validation of the TcsL-SEMA6A interaction interface. The cytotoxicity of wild-type TcsL and TcsL 4mut variant with four mutated interaction interface residues (C1433D-I1434K-A1486S-Y1596R; shown in A) was assessed in Vero cells. (D) Validation of SEMA6A M109 as a critical interacting residue with TcsL. SEMA6A/SEMA6B double knockout cells were infected with 3xFLAG-tagged wild-type SEMA6A or M109D mutant and assayed for sensitivity to TcsL. Protein expression levels were confirmed by western blotting (right). See also Figure S2 , Figure S3 , Figure S4 , Figure S5 .

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Comparison, Binding Assay, Biomarker Discovery, Variant Assay, Residue, Double Knockout, Infection, Mutagenesis, Expressing, Western Blot

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: Conservation of SEMA6A and TcsL interface residues in semaphoring and large clostridial toxin families, related to Figure 6 A, Sequence alignment of SEMA6 family proteins. Residues conserved in all four SEMA6 family proteins are denoted in light blue. SEMA6A residues forming contacts with TcsL are indicated as red circles, and those interacting with Plexin A2 are indicated as blue circles. The two TcsL-interacting residues that differ between SEMA6A/SEMA6B and SEMA6C/SEMA6D are colored orange. Secondary structure elements with numbered beta-propeller blades are shown below the alignment. (B) The receptor-binding surface of TcsL and TcdB is highly divergent between all clostridial toxins. Partial sequence alignment of six known large clostridial toxins. Secondary structure elements and the consensus sequence (at least 4/6 identical residues) are shown above the alignment. Amino acids are colored based on their biophysical properties (ClustalX coloring) if at least 4/6 residues are similar in each column. Red boxes indicate interface residues in TcsL/SEMA6A or TcdB/Fzd2 complexes. The gray box highlights the evolutionarily divergent beta sheet in the receptor-binding interface. (C) Alignment entropy was calculated as a 20-aa moving window along the alignment of six known clostridial toxins. Red bars indicate the location of TcsL/SEMA6A interface residues.

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Sequencing, Binding Assay

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet: TcsL and TcdB bind different host receptors through the same interface region (A) Sequence alignment entropy in large clostridial toxin family shown as a rainbow spectrum on the TcdB full-length cryo-EM structure (PDB ID: 6OQ5 ) ( Chen et al., 2019 ). Entropy was calculated as a 10-aa moving window. The receptor-binding surface is indicated. (B) SEC profiles and SDS-PAGE analysis of FZD7-TcdB 1285–1804 (top), SEMA6A-TcsL 1285–1804 (middle), and FZD7-TcsL 1285–1804 (bottom). SEC fractions used for SDS-PAGE analysis are highlighted with an asterisk. (C) Sequence alignment between TcsL and TcsB. TcsL residues interacting with SEMA6A are highlighted in pink and TcdB residues contacting FZD2 are highlighted in orange. Black dots denote the 15 mutations introduced in TcsL (FBD) 1285–1804 variant that resulted in shifting the TcsL binding specificity from SEMA6A to FZD7. (D) SEC profiles and SDS-PAGE analysis of FZD7-TcsL (FBD) 1285–1804 (TcsL variant with a TcdB-like binding interface) (top) and SEMA6A-TcsL (FBD) 1285–1804 (bottom). SEC fractions used for SDS-PAGE analysis are highlighted with an asterisk. See also Figure S6 .

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Sequencing, Cryo-EM Sample Prep, Binding Assay, SDS Page, Variant Assay

Journal: Cell

Article Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins

doi: 10.1016/j.cell.2020.06.005

Figure Lengend Snippet:

Article Snippet: Mouse Sema6A-Fc and Sema6c-Fc expression constructs were a gift from Woj Wojtowicz (Addgene plasmids 72163 and 72167, respectively) and human SEMA6A was gene-synthesized (GeneArt).

Techniques: Plasmid Preparation, Recombinant, Expressing, Transfection, Luciferase, Cell Viability Assay, CRISPR, Knock-Out, Software

Journal: Nucleic Acids Research

Article Title: Locus-specific transcription silencing at the FHIT gene suppresses replication stress-induced copy number variant formation and associated replication delay

doi: 10.1093/nar/gkab559

Figure Lengend Snippet: Altered RNase H1 expression does not change genome-wide APH-induced CNV frequencies in HF1 fibroblasts. ( A ) RNASEH1 RNA levels were determined by qRT-PCR for HF1 fibroblast cell clones carrying shRNA constructs that were either scrambled sequence controls or specific to RNASEH1 . Expression decreased for each of two independent knockdown clones upon shRNA induction with 48 h of doxycycline. Results are the fold change relative to the untreated scrambled control and are the mean ± standard deviation of triplicate measurements. ( B ) RNASEH1 RNA knockdown persists through 48 h of doxycycline treatment followed by 72 h of APH treatment. (A) and (B) are from different experiments. ( C ) Western blots of the cell clones in (A) revealed parallel changes in RNASEH1 protein levels. Alpha-tubulin and actinin served as loading controls for experiments conducted at different times. ( D ) Genome-wide CNV formation was determined by microarray analysis of two cell clones expanded from RNASEH1 knockdown populations ± APH treatment, all treated with doxycycline. Results are expressed as the mean ± 95% confidence interval of the CNV rate (the number of de novo CNVs divided by the number of cell clones examined). ( E – G ) similar to (A), (B) and (C), showing increased RNase H1 RNA and protein levels for two independent clones that overexpressed RNase H1 in response to doxycycline, as compared to an empty expression vector. ( H ) similar to (D), showing CNV analysis for two clones with RNase H1 overexpression.

Article Snippet: Proteins were transferred to PVDF membranes and incubated with 1:500 anti-RnaseH1 mouse monoclonal antibody (Sigma, WH0246243M1) or 1:1000 anti-Alpha Actinin rabbit monoclonal antibody (Cell Signaling Technology, 6487T) in 5% milk and TBST overnight at 4°C.

Techniques: Expressing, Genome Wide, Quantitative RT-PCR, Clone Assay, shRNA, Construct, Sequencing, Knockdown, Control, Standard Deviation, Western Blot, Microarray, Plasmid Preparation, Over Expression

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A EHMT1 interacting proteins identified by mass spectrometric analysis with details indicating coverage and peptide score. B Sequential IP in HEK293 cells demonstrating EHMT1, EHMT2, and LMNB1 are a part of the same complex. C LMNB1 interacts with EHMT1 via SET domain. Recombinant GST or GST-LMNB1 was incubated with Ni-NTA bound His-EHMT1 SET protein. Post washing eluents were loaded for immunoblotting using GST or His antibody. Recombinant pure proteins GST-LMNB1 (lane 1), GST (lane 2), EHMT1-SET (lane 3) were used as controls. D Venn diagram showing unique and overlapping reads obtained from EHMT1 and LMNB1 ChIP-Sequencing. E Composite profile of EHMT1 and LMNB1 read density around the transcription start site (TSS). F Genomic distribution of EHMT1 and LMNB1 peaks. The majority of binding sites obtained were enriched in an intronic region or distal regions from a gene. G, H Representative figure showing normalized ChIP-seq read density (above 1.5-fold over expected) of EHMT1 and LMNB1 in 1MB bin for chromosome 1 & 9.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Recombinant, Incubation, Western Blot, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A EHMT1 interacts with EHMT2, LMNB1, and HP1. Whole cell lysates from fetal HDFs were subjected to IP reaction using EHMT1 or LMNB1 antibody. Subsequently, western blot was performed using the IPed material to detect EHMT1, EHMT2, LMNB1, and HP1. ASH2L did not show any interaction with EHMT1 and LMNB1 thus acted as a negative control. B EHMT1 interacts with LMNB1 and EHMT2 via SET domain. HEK293 cells were transfected with pEGFP-Ankyrin (pEGFPC1-ANK) or pEGFPC1-SET domains of EHMT1 to determine domain specific association with LMNB1. Cell extracts were subjected to IP using GFP-antibody and the bound complexes were then analyzed by immunoblotting using LMNB1, EHMT2, and GFP antibodies. HEK-293 whole cell extract represents 1% input. pEGFPC1 empty vector transfected HEK293 or untransfected HEK293 cells were used as control reactions. Arrows indicate specific band. C Coomassie stained SDS-PAGE gel showing 6X His EHMT1-SET purified protein used for methyltransferase assays. D, E, F Gene Ontology (GO) analysis of EHMT1 and LMNB1 bound genes. Representative figure showing enriched GO terms for EHMT1 ( D ), LMNB1 ( E ) and EHMT1 and LMNB1 ( F ) co-bound genes. The length of the bar (y-axis) denotes total genes falling within GO term. G Circos plot showing genome wide peak density of EHMT1 (green) and LMNB1 (red).

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Negative Control, Transfection, Plasmid Preparation, Staining, SDS Page, Purification, Genome Wide

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Increasing concentrations of LMNB1-GST showed a greater degree of methylation by EHMT1-SET. Methyltransferase assay was performed using a fixed concentration of recombinant 6X His EHMT1-SET as an enzyme source, and SAM as a methyl group donor. Recombinant GST-LMNB1 (4.5 ng and 9 ng) and Histone H3 peptide (10 ng) were used as substrates in the assay. Histone H3 peptide was used as a positive control. The mean relative fluorescence unit (RFU) values represented in the graph were obtained after subtracting the values for controls EHMT1-SET only, SAM only, LMNB1-GST (4.5 ng) with those of the LMNB1-GST (4.5 ng and 9 ng) and Histone H3 (10 ng). B Coomassie stained SDS-PAGE gel showing 6X His LMNB1-CT purified protein used for methyltransferase assays. C mWasabi expression in fetal HDFs transduced with Wt.LMNB1 and K417A-LMNB1 mutant construct. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented. D MFI for mWasabi expression in cells transfected with Wt.LMNB1 and K417A-LMNB1 mutant constructs. Each biological replicate is represented as spread of technical replicates around the mean value. E MFI for mWasabi expression in cells transfected with Wt.LMNB1 and K417A-LMNB1 mutant constructs. MFI has been represented as center vs periphery of the nuclei. Individual biological replicates are plotted with the mean values. F Quantitation for percentage distorted nuclei in cells transfected with K417A-LMNB1 mutant construct compared to Wt.LMNB1 construct. Individual biological replicates are plotted with the median. G Immunostaining for LMNA/C in fetal HDFs transduced with Wt.LMNB1 and K417A-LMNB1 mutant construct. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Methylation, Concentration Assay, Recombinant, Positive Control, Fluorescence, Staining, SDS Page, Purification, Expressing, Transduction, Mutagenesis, Construct, Transfection, Quantitation Assay, Immunostaining

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Western blot probed with the anti-Methyl-K antibody. Lanes to the left of the ladder are control reactions containing 2 μg LMNB1-CT + 50 μM SAM (lane 1) or 2 μg of EHMT1-SET/EHMT2-SET + 2 μg LMNB1-CT (lane 2) and 2 μg LMNB1-CT only (lane 3). Lanes to the right of the ladder are methyltransferase reactions containing 1, 2 and 4 μg of LMNB1-CT and EHMT1-SET/EHMT2-SET along with 50 μM SAM. Lower panel: Coomassie stained gel representing all the reactions mentioned above. C, D Nuclear lysates from HEK-293 were immunoprecipitated with anti-Methyl-K or anti-LMNB1 antibody followed by western blotting with anti-Pan H3, anti-LMNB1 and anti-Methyl-K antibodies. Asterisk indicates the band of interest. E Reduced methylation of LMNB1 by EHMT1 and EHMT2 upon single amino acid substitution from lysine (RKR) to alanine (RAR). Mutant LMNB1 peptides were synthesized and subjected to methyltransferase reaction containing EHMT1-SET and EHMT2-SET with SAM as a methyl donor. H3 peptide was used as a positive control for the reaction. F Immunostaining for LMNB1 in fetal HDFs transfected with Wt.LMNB1 and K417A-LMNB1 constructs. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Staining, Immunoprecipitation, Methylation, Mutagenesis, Synthesized, Positive Control, Immunostaining, Transfection, Construct

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for EHMT1 and EHMT2 in fetal HDFs upon knock down of EHMT1 and EHMT2. GAPDH was used as a loading control. B, C Relative protein levels of EHMT1 (B) and EHMT2 (C) upon knockdown. Each biological replicate is represented as spread of technical replicates around the mean value. D Percentage of distorted nuclei increases in human fibroblasts transduced with shEHMT1 and shEHMT2 virus compared to UT or shCnt. (n=3), For UT, (n=43); shCnt, (n=23); shEHMT1, (n=18); shEHMT2, (n=20) nuclei counted. shCnt vs shEHMT1, **p=0.0048; shCnt vs shEHMT2, #p=0.0153. (Kruskal-Wallis test, post-hoc test: Dunn’s multiple comparison test)

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Transduction

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for LMNB1 and LMNA/C in fetal HDFs transduced with shEHMT1 and shEHMT2 virus. Untransduced (UT) and control shRNA (shCnt) were used as controls. B, C Fetal HDFs were transduced with shCnt, shEHMT1 and shEHMT2 virus. Post transduction cells were immunostained for EHMT1 or EHMT2 and co-stained for NL using LMNB1 antibody. Distortion of nuclear architecture was seen upon loss of EHMT1 and EHMT2 proteins. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented. D Relative expression of LMNA and LMNB1 in fetal HDFs upon EHMT1 and EHMT2 knockdown compared to UT/shCnt cells. (n=3) For LMNB1: UT vs shEHMT1, ***p<0.0001 and UT vs shEHMT2, ***p<0.0001. (One Way ANOVA, post-hoc test: Bonferroni’s multiple comparison test).

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Transduction, shRNA, Staining, Expressing

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for H3K9me2 and H3K9me3 in UT, shCnt, shEHMT1 and shEHMT2 transduced HDFs. GAPDH was used as loading control. B Quantification of H3K9me2 upon EHMT1 and EHMT2 knockdown. Each biological replicate is represented as spread of technical replicates around the mean value. C Immunostaining for H3K9me2 co-stained for nuclear lamina using LMNB1 antibody in fetal HDFs transduced with shCnt, shEHMT1 and shEHMT2 virus. (Scale bar: 20μm). Insets are zoomed images of cells in the merge. D H3K9me2 immunostaining in fetal HDFs transduced with shEHMT1.1 (lentivirus) and shEHMT2.1 (retrovirus). (Scale bar: 20μm). E MFI profile (Centre to periphery) with standard deviation for all the measured nuclei of shCnt, shEHMT1 and shEHMT2. F Immunostaining for H3K9me2 in fetal HDFs treated with or without BIX 01294 (1μM) for 48 h. (Scale bar: 20μm). G MFI plot for H3K9me2 staining in fetal HDFs treated with or without BIX 01294 (1μM) for 48 h. Each biological replicate is represented as spread of technical replicates around the mean value. H Western blot for LMNB1 and H3K9me2 on cells treated with BIX 01294 or vehicle control. BIX 01294 treatment resulted in a reduction of H3K9me2 levels without altering LMNB1 expression. I In vitro methylation assay was performed using recombinant LMNB1-CT and EHMT1-SET or EHMT2-SET domain in the presence or absence of BIX 01294. H3 peptide was used as a positive control. J TEM image for fetal HDFs treated with BIX 01294. BIX 01294 treatment did not affect the peripheral heterochromatin distribution. (Scale bar: 1μm) K MFI for H3K9me2 staining in cells transfected with Wt.LMNB1 and K417A-LMNB1 constructs. Each biological replicate is represented as spread of technical replicates around the mean value. L Western blot showing expression of endogenous LMNB1 (top panel lower band) and Wasabi tagged LMNB1 (top panel upper band). The lysates from cells transfected with Wt.LMNB1 and K417A-LMNBI were probed with the LMNB1 antibody. The lysate was also probed with H3K9me2 antibody and H3 (loading control). M MFI for H3K9me2 staining in cells transfected with Wt.LMNB1 and K417A-LMNB1 constructs. MFI has been represented as center vs periphery of the nuclei. Each biological replicate is represented as spread of technical replicates around the mean value.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Immunostaining, Staining, Transduction, Standard Deviation, Expressing, In Vitro, Methylation, Recombinant, Positive Control, Transfection, Construct

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A MFI for H3K9me2 staining in fetal HDFs transduced with shEHMT1 and shEHMT2. MFI has been represented as center vs. periphery of the nuclei and compared with respect to shCnt cells. Each biological replicate is represented as spread of technical replicates around the mean value. B, C, D Fetal HDFs were transduced with shCnt, shEHMT1 and shEHMT2 virus. TEM was performed to visualize the heterochromatin in EHMT1 and EHMT2 knockdown cells. Peripheral heterochromatin was intact at the NP beneath the NL in control cells while this distribution was drastically altered upon knockdown of EHMT1 and EHMT2. (Scale bar: 1μm). Arrows indicate the heterochromatin regions. E Immunostaining for H3K9me2 in fetal HDFs expressing Wt.LMNB1 or K417A-LMNB1 mutant plasmids. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented. F, G Fetal HDFs transfected with Wt.LMNB1 and K417A-LMNB1 plasmids were processed for electron microscopy. Cells transfected with Wt.LMNB1 plasmid showed intact peripheral heterochromatin while K417A-LMNB1 transfected cells showed loss of heterochromatin with nuclear envelope breaks. (Scale bar: 1μm). Arrows indicate the zoomed area presented in the inset below. H qRT-PCR to validate expression of EZH2 in UT, shEHMT1 and shEHMT2 transduced HDFs. (n=3), UT vs. shEHMT1 (**p=0.0067), UT vs. shEHMT2 (**p=0.0021). (One sample t-test, two-tailed). I Immunostaining for H3K27me3 mark and HP1 protein in fetal HDFs transduced with shCnt, shEHMT1, and shEHMT2. Knockdown of EHMT1 and EHMT2 leads to a reduction in the H3K27me3 mark, which corroborates with decreased Ezh2 expression while HP1 expression was reduced only in shEHMT1 cells. (Scale bar: 20μm)

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Staining, Transduction, Immunostaining, Expressing, Mutagenesis, Transfection, Electron Microscopy, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Heat map for differential expression of (A) all genes and (B) chromatin modifiers obtained from RNA-seq analysis of shEHMT1 or shEHMT2 compared to shCnt transduced HDFs. Representative genes that were altered similarly or distinctly in EHMT1 vs EHMT2 are indicated from few clusters.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Heat map demonstrating differential expression of age related genes in EHMT1 and EHMT2 depleted fibroblasts. Representative genes that were altered similarly or distinctly in EHMT1 vs EHMT2 are indicated from few clusters. B. Validation for differential expression of candidate genes obtained from RNA-Seq analysis by semi-quantitative PCR. C. Validation for differential expression of candidate genes obtained from RNA-Seq analysis by qRT-PCR (n=3), UT vs FGFR1, p=0.05; UT vs CDKN2B, p= 0.0058; UT vs FOXM1, p=0.0018 One sample t-test, two tailed. D. Immunostaining for H3K9me3, H3K27me3 and HP1 in fetal, 18Y old and 65Y old HDFs followed by confocal imaging. (Scale bar: 20μm) E. Western blot analysis for H3K9me3 and H3K27me3 in fetal, 18Y and 65Y old HDFs.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Immunostaining, Imaging, Western Blot

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for EHMT1, EHMT2 and GAPDH in HDF cell lysates derived from various age groups. B , Quantification of EHMT1 protein in HDFs of indicated age groups. One Sample t test (n=3). For fetal vs 65 (*p<0.05), Fetal vs 18Y and Fetal vs 31Y are not significant. C , Quantification of EHMT2 protein expression in HDFs of indicated age groups. One Sample t test (n=6). For Fetal vs 18Y (***p=0.0004), and Fetal vs 65Y (***p=0.0002). D Immunostaining for EHMT1, EHMT2 and Pan H3 in fibroblasts from indicated age groups (Scale bar: 20μm). E, F Western blot and immunostaining (Scale bar: 20μm) analysis for H3K9me2 in indicated age groups. G Representative TEM images for nuclei of 18Y, 31Y, 40Y and 65Y HDFs. Arrows indicate peripheral heterochromatin. Inset is the zoomed version of the same images. (Scale bar: 1μm). H Western blot analysis for LMNA/C and LMNB1 and in various age groups HDFs. GAPDH was used as an internal control. I Quantification of LMNB1 protein expression in HDFs of indicated age groups. One sample t test (n=6). For Fetal vs 18Y (**p=0.0069) and Fetal vs 65Y (**p=0.0016). J,K,L Reduced levels of methylated LMNB1 during physiological aging. Nuclear extracts from indicated age groups were subjected to IP using LMNB1 antibody. IPed material was divided into halves, Blot 1 (left,J) was probed with anti LMNB1 antibody, blot 2 was probed with anti-Methyl-K (middle, K) antibody. Antimethly-K blot was reprobed with LMNB1 antibody to demonstrate the methyl band indicated is indeed LMNB1 (Right, L). 80 μg of fetal HDFs derived nuclear lysate was used as input control.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Derivative Assay, Expressing, Immunostaining, Methylation

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A-C. MFI (Centre to periphery) with standard deviation for H3K9me2 staining in all the measured nuclei of fetal, 18Y and 65Y old HDFs. D. MFI for H3K9me2 staining in fetal, 18Y and 65Y old HDFs. MFI has been represented as center vs periphery. Individual biological replicates are plotted with mean values. E Relative expression of EHMT1 and EHMT2 at the mRNA level in fetal, 18Y and 65Y old HDFs. (n=3), For EHMT1: Fetal vs 65Y, *p=0.0219; For EHMT2: Fetal vs 65Y, #p=0.0219. (Kruskal-Wallis test, post-hoc test: Dunn’s multiple comparison test). F Western blot for EHMT1 and EHMT2 in 18Y and 65Y old HDFs treated with or without proteasomal degradation inhibitor MG132 (10μM for 6 h). G Quantification of EHMT1 and EHMT2 protein levels in 18Y old HDFs treated with or without MG132 treatment. (n=3), for 18Y EHMT1: UT vs MG132, p=0.5989, ns; For 18Y EHMT2: UT vs. MG132 (*p=0.0154). (One sample t-test, two-tailed). H-I. Cell lysates prepared from human fibroblasts of indicated age groups were subjected for IP using EHMT1/EHMT2 antibody. IPed material was analyzed by immunoblotting using LMNB1 and LMNA/C antibodies. 30 μg of fetal cell lysate was used as input control. Dotted lines indicate that different exposures were used for Input and IP of the same western blot.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Standard Deviation, Staining, Expressing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Immunostaining using V5 or Flag antibodies along with LMNB1 in 65Y HDFs transfected with V5-EHMT1 and Flag-EHMT2 overexpression constructs. Immunostaining using H3K9me2 antibody along with LMNB1 in 65Y HDFs transfected with V5-EHMT1 and Flag-EHMT2 overexpression constructs. (Scale bar: 20μm) Arrows indicate the cells zoomed in the far-right image presented. C, D, E 65Y Old HDFs overexpressing EHMT1 and EHMT2 were processed for electron microscopy. Overexpression of EHMT1 and EHMT2 causes restoration of peripheral heterochromatin in old cells compared with control cells (Scale bar: 1μm). Arrows indicate the area zoomed and presented in the inset format.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Immunostaining, Transfection, Over Expression, Construct, Electron Microscopy

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A-B. Quantitation of LMNB1 expression in aged HDFs upon overexpression of EHMT1 and EHMT2. Overexpression of EHMT1 significantly increased the LMNB1 expression while EHMT2 transfected cells did not show any change compared to untransfected cells. Biological replicates of V5-EHMT1 and Flag-EHMT2 expressing cells were plotted with the mean values. C, D,E. Old HDFs expressing Wt. LMNB1 + V5-EHMT1, K417A-LMNB1 + V5-EHMT1 and K417A-LMNB1 + Flag-EHMT2 were stained with H3K9me2 antibody. Mutation at lysine 417 position of LMNB1 affects the overall distribution H3K9me2 and morphology (Scale bar: 20μm). Arrows indicate the cells zoomed in the far right image presented.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Quantitation Assay, Expressing, Over Expression, Transfection, Staining, Mutagenesis

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Knockdown of EHMT1 and EHMT2 reduces cell proliferation. An equal number of HDFs were seeded and transduced with shCnt, shEHMT1 and shEHMT2 virus. Forty-eight hours post transduction, the number of cells in the culture was counted over a period of four days as indicated. (n=3), for shEHMT1, Day3: shCnt vs. shEHMT1 (**p=0.0015); Day4: shCnt vs. shEHMT1 (***p<0.0001); for shEHMT2, Day1: shCnt vs. shEHMT2 (***p=0.0002), Day2: shCnt vs. shEHMT2 (***p<0.0001), Day3: shCnt vs. shEHMT2 (***p<0.0001), Day4: shCnt vs. shEHMT2 (***p<0.0001). (Two-way ANOVA, post-hoc: Tukey’s multiple comparison test) C Quantification of cell cycle analysis. Increase in sub-G1 population an indication of cell apoptosis upon EHMT2 knockdown. For shCnt, (n=3), shEHMT1, (n=2) and shEHMT2, (n=2) SubG1: shCnt vs. shEHMT2 (**p=0.0011) (Two-way ANOVA, post-hoc: Tukey’s multiple comparison test). D, E Equal number of HDFs were seeded and transduced with shCnt, shEHMT1 and shEHMT2 virus. β-Galactosidase (senescence) assay was performed to monitor the cellular senescence in cultures. Fold change in an average number of senescent cells per field was quantitated. F, G TRAP assay to detect telomerase activity in indicated age groups as well as in shCnt, shEHMT1 and shEHMT2 transduced cells. Fetal, 18Y and shCnt fibroblast lysates were heat inactivated (HI) as a negative control for the assay. Telomerase activity was reduced upon knockdown of EHMT1 and EHMT2 as well as during physiological aging from fetal to 65Y old cells.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Transduction, Cell Cycle Assay, TRAP Assay, Activity Assay, Negative Control