Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) Reporter architecture and characterization of its integration site. A reporter containing EGFP driven by the elongation factor 1α promoter (pEF), preceded by five Tet Operator sites (5xTetO), and a hygromycin resistance gene driven by the SV40 early promoter > 5kb upstream was site-specifically integrated into the genome of HEK293 cells. UCSC browser snapshot of ENCODE H3K4me3, H3K36me3, and H3K9me3 ChIP-seq tracks shows the euchromatic integration site. Two replicates of ChIP (colored) and input (gray) signals are overlaid. The H3K9me3 track on a KRAB-Znf gene cluster is shown as a reference for an H3K9me3-enriched region. (B) Schematic diagram of the inducible reporter system for studying heterochromatin. The rTetR-fused heterochromatin protein is stably co-expressed with the reporter and binds to TetO sites upon addition of doxycycline (Dox), and is released upon Dox removal. Analysis of transcriptional silencing efficiency and deposition and spreading of heterochromatin marks upon Dox supplementation and its removal after silencing establishment enables temporal evaluation of heterochromatin establishment, spreading, and maintenance. Red bars A–C denote amplicons used for qPCR analysis. (C) Artificial recruitment of HP1α leads to reporter silencing. GFP signal was measured by flow cytometry at different time points after rTetR-HP1α recruitment to the reporter. Dox was removed after 14 days of recruitment, and GFP signal measurement continued for an additional 15 days. The black line indicates the gating threshold to distinguish between GFP-positive (GFP+, yellow shading) and GFP-negative (GFP-, gray shading) cell populations. Dox-treated (blue) and control (gray) samples are shown. Right: Quantification of three biological replicates at indicated times. SD is plotted, but is too small to show for most points. (D) HP1α tethering induces H3K9me3 deposition over EGFP and the hygromycin resistance gene. H3K9me3 ChIP-qPCR was performed 3 days after HP1α recruitment. H3K9me3 ChIP-qPCR analysis was performed with and without Dox at three regions over the reporter as indicated in panel B. Dots correspond to independent biological replicates; bars indicate mean and SD. (E) SetDB1 is required for reporter silencing upon HP1α tethering. SetDB1 was knocked out in HEK293 cells that stably express both the reporter and rTetR-HP1α. Two single clones were isolated and validated by PCR (data not shown) and western blot (top). HP1α recruitment to the reporter was performed in SetDB1-WT cells and the two SetDB1-KO clones. GFP signals were measured by flow cytometry at day 5 (orange) and day 14 (green) after Dox treatment, or without Dox (gray). (F) SetDB1 knockdown decreases H3K9me3 deposition upon HP1α tethering. Control siRNA and siRNA against SetDB1 were transfected into cells stably expressing both the reporter and rTetR-HP1α. RNAi efficiency was determined 5 days post-siRNA transfection (left). HP1α was recruited to the reporter two days after siRNA transfection, and H3K9me3 ChIP-qPCR was performed 3 days later. Two regions are detected as described above. Dots correspond to independent biological replicates; bars indicate mean and SD.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: ChIP-sequencing, Stable Transfection, Flow Cytometry, Control, Clone Assay, Isolation, Western Blot, Knockdown, Transfection, Expressing

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) Artificial recruitment of SetDB1 leads to efficient silencing of the reporter. Left: Schematic diagram of the inducible reporter system. Right: rTetR-SetDB1 was recruited to the reporter upon Dox supplementation in the medium, and GFP signal was measured by flow cytometry at different time points. After 14 days of Dox treatment, Dox was removed from the medium, and flow cytometry measurements continued for an additional 15 days. Data represent the mean of 3 biological replicates. SD are plotted but are too small to be visible. (B-C) SetDB1 tethering induces de novo H3K9me3 and HP1 deposition along the reporter sequence. H3K9me3 ChIP-qPCR was performed after 3 and 14 days of SetDB1 recruitment (B). HP1α ChIP-qPCR was performed after 14 days of SetDB1 recruitment (C). The locations of amplicons along the reporter are indicated in . Dots represent independent biological replicates; bars indicate the mean ± SD.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Flow Cytometry, Sequencing

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) SetDB1 physically interacts with HP1α. EGFP-SetDB1, purified from HEK293T cells using wash conditions of different stringency (150 mM–1 M NaCl), was used to pull down HP1α-Flag-His purified from E. coli . Proteins were detected using anti-GFP and anti-Flag antibodies. (B) The chromodomain (CD), but not the chromoshadow (CSD) domain, of HP1α shows a strong interaction with SetDB1. A diagram of HP1α protein structure is shown at the top. Full-length (FL) or truncated fragments of EGFP-HP1α were transiently co-expressed with Flag-SetDB1 in HEK293T cells. Co-immunoprecipitation was performed using GFP nanotrap beads. KAP1, a known interactor of HP1α CSD, was detected for comparison. (C) HP1α mutants that disrupt the HP1-H3K9me3 interaction also impair HP1-SetDB1 interaction. WT or mutant EGFP-HP1α was transiently co-expressed with Flag-SetDB1 in HEK293T cells, followed by co-immunoprecipitation using GFP nanotrap beads and western blot analysis. (D) H3K9me3 peptide competes with SetDB1 for HP1α binding. EGFP-SetDB1 was purified from HEK293T cells using GFP nanotrap beads. HP1α-Flag-His purified from E. coli and synthesized histone peptides (methylated or unmethylated) were incubated with the bead-coupled EGFP-SetDB1. HP1α was detected in input, IP, and flowthrough fractions by western blot.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Purification, Immunoprecipitation, Comparison, Mutagenesis, Western Blot, Binding Assay, Synthesized, Methylation, Incubation

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: WT or W174A mutant EGFP-HP1α and Flag-SetDB1 were transiently co-expressed in HEK293T cells and co-immunoprecipitated using GFP nanotrap beads. The indicated proteins were detected by western blot. Cells co-expressing EGFP and Flag-SetDB1 were used as a negative control.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Negative Control

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: Biotin-conjugated H3K9me3 peptide was incubated with cell lysate containing EGFP-HP1α and its respective mutants. Pulldown was performed using streptavidin beads, and EGFP-HP1α signal was detected by western blot. EGFP and EGFP-SetDB1 were used as negative controls.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Incubation, Western Blot

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) Two different regions (aa 410-666 and aa 666-1291) are predicted to interact with HP1α. Full-length (FL) or truncated EGFP-SetDB1 and HP1α-Flag were co-expressed in HEK293T cells. Co-immunoprecipitation was performed using GFP nanotrap beads, and western blot was carried out to detect the immunoprecipitating proteins. Numbers indicate the expressed amino acid stretches. (B) AlphaFold2 predicts two motifs on SetDB1 that interact with the HP1α chromodomain. Full-length HP1α was submitted to AlphaFold2-Multimer along with two different SetDB1 regions (aa 410-666 and aa 666-1291). The PAE (predicted aligned error) files show five different interaction models generated by AlphaFold2-Multimer for each prediction. The two SetDB1 regions with the lowest PAE values are labeled as motif 1 and motif 2. (C) The predicted structures formed by HP1α-SetDB1 interactions show high similarity to the reported HP1-H3K9me2 interaction. The HP1 chromodomain is shown in blue, with relevant residues forming the binding pocket in green (left, reported HP1-H3K9me2 interaction ) or in green with relevant residues marked in pink (middle and right, predicted HP1α-SetDB1 interactions). Histone H3, SetDB1 motif 1, and motif 2 are shown in yellow (main chain) and red (side chains).

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Immunoprecipitation, Western Blot, Generated, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) Domain architecture of SetDB1. Two HP1α-interacting motifs, predicted by AlphaFold2-Multimer, are highlighted, along with their corresponding sequences. (B) Sequence alignment of the histone H3 tail and the two identified SetDB1 motifs. H3K9 and the corresponding lysine residues in SetDB1 are shown in red. (C) HP1α interacts exclusively with methylated SetDB1 peptides in vitro . HP1α-Flag-His, purified from E. coli , was incubated with biotin-conjugated, lysine-trimethylated (Me3) or unmethylated (Un-Me) histone H3 or SetDB1 peptides. Pulldown was performed using streptavidin beads, and HP1α-Flag-His was detected by western blot. The negative control contained no peptide in the pulldown assay. (D) SetDB1 HMM mutants lose trimethylation and HP1 interaction. HP1α-Flag-His, purified from E. coli , was incubated with cell lysates from HEK293T cells expressing EGFP-SetDB1 (WT or mutants) and immunoprecipitated using GFP nanotrap beads. Cells expressing EGFP alone served as a negative control. HMm indicates a mutant with lysine-to-alanine mutations at positions 489, 490, and 1162. H3K9me3 antibody was used to detect trimethylated SetDB1.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Sequencing, Methylation, In Vitro, Purification, Incubation, Western Blot, Negative Control, Expressing, Immunoprecipitation, Mutagenesis

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: Sequence alignments of SetDB1 in different species ( Homo sapiens (Hs), Bos taurus (Bt), Mus musculus (Mm), Gallus gallus (Gg), Xenopus laevis (Xi), Danio rerio (Dr), Bombyx mori (Bm), Drosophila melanogaster (Dm) ) were performed using NCBI-COBALT. A schematic of positions colored by percentage identity is shown. Histograms reflect conservation and consensus. SetDB1 motif 1 and motif 2 are marked by red bars. The regions containing motif 1 and motif 2 and their flanking sequences are magnified (bottom). Images are based on alignments edited in Jalview .

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) EGFP-SetDB1 and mutants were expressed and immunoprecipitated from HEK293T cells. Endogenous HP1α and MPP8 were detected by western blot. (B) EGFP-SetDB1 was co-expressed with MPP8-Flag or HP1α-Flag in HEK293T cells and immunoprecipitated using GFP nanotrap beads. Overexpressed HP1α and MPP8 were detected by Western blot using an anti-Flag antibody.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) SetDB1 K490 and K1162 can be tri-methylated in vivo . EGFP-SetDB1 was purified from HEK293T cells, digested with thermolysin, and analyzed by tandem mass spectrometry. Representative MS2 spectra of peptides with trimethylation at K490 (left, VAKKSTS, 424.7452 m/z) and K1162 (right, VAVKSTRGFA, 560.8273 m/z) are shown. The b and y ion peaks corresponding to the trimethylated peptides are labeled. The “-C3H9N” fragment indicates trimethylation. Carbamylation is an artificial modification occurring during thermolysin digestion. (B) SetDB1 methylates HMM motifs in vitro . EGFP-SUMO EU -SetDB1 was expressed in HEK293T cells, immunopurified using GFP nanotrap beads, and SetDB1 was eluted by SUMO protease SENP EUB cleavage (left). Quantification of the methyltransferase activity of purified SetDB1 on histone H3 tail or SetDB1 HMM-containing peptides was performed using a commercial kit. Methylation of H3K9 was quantified using an HRP-conjugated secondary antibody-color development system at OD450 (right). Dots represent independent biological replicates; bars indicate mean ± SD. (C) SetDB1 lacking methyltransferase activity cannot efficiently interact with HP1α. EGFP-SetDB1-WT or methyltransferase-dead mutants were transiently expressed in SetDB1-knockout HEK293T cells. Cell lysates were incubated with HP1α-Flag-His purified from E. coli , followed by co-immunoprecipitation using GFP nanotrap beads and western blot analysis. EGFP alone was used as a negative control.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Methylation, In Vivo, Purification, Mass Spectrometry, Labeling, Modification, In Vitro, Activity Assay, Knock-Out, Incubation, Immunoprecipitation, Western Blot, Negative Control

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: WT or mutant EGFP-SUMO EU -SetDB1 was expressed and purified from HEK293T cells. SetDB1 was eluted from the beads using SUMO protease SENP EUB (left). Quantification of the methyltransferase activity of purified SetDB1 on histone H3 peptide was performed using a commercial kit. The amount of methylated H3K9 was quantified using an HRP-conjugated secondary antibody and color development system under OD450 (right). Dots represent independent biological replicates; bars indicate the mean ± SD.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Mutagenesis, Purification, Activity Assay, Methylation

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: EGFP-SetDB1-WT or methyltransferase-dead mutants (H1224K or C1226A) were transiently expressed in HEK293T cells that endogenously express SetDB1. Cell lysates were incubated with HP1α-Flag-His purified from E. coli , followed by co-immunoprecipitation using GFP nanotrap beads and western blot analysis. EGFP alone was used as a negative control. Both EGFP-SetDB1 methylation and its interaction with HP1α were strongly reduced in the mutants.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Incubation, Purification, Immunoprecipitation, Western Blot, Negative Control, Methylation

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) SetDB1-HMm stabilizes the SetDB1 cofactor ATF7IP. WT or HMm mCherry-SetDB1 was stably expressed in SetDB1-KO HEK293 cells. Proteins were detected from total cell lysates by Western blot using antibodies against SetDB1, ATF7IP, and Tubulin. (B) SetDB1-HMm is present in the nucleus. WT or HMm mCherry-SetDB1 was stably expressed in SetDB1-KO HEK293 cells. Immunofluorescence was performed using an antibody against SetDB1. Nuclei were stained with DAPI.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Stable Transfection, Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) Schematic diagram of cells engineered to analyze the effects of HMM mutation (HMm) on SetDB1 (top) and HP1α (bottom) induced heterochromatin. For SetDB1 tethering, WT or HMm rTetR-SetDB1 was stably expressed in cells harboring the reporter. For HP1 tethering, WT or HMm mCherry-SetDB1 was stably expressed in SetDB1-KO cells expressing the reporter and rTetR-HP1α. (B) Reporter silencing upon HP1α and SetDB1 tethering is unaffected by SetDB1-HMM mutation. GFP signal was measured by flow cytometry upon SetDB1 or HP1α recruitment to the reporter over different periods (4, 7, and 14 days). The histograms (left) show results from representative single clones. The black line indicates the gating threshold to distinguish between GFP-positive (GFP+, yellow shading) and GFP-negative (GFP-, gray shading) cell populations. Dox-treated (purple/blue/orange) and no-Dox control (gray) samples are shown. Bar graphs (right) show the quantitative results from different clones. Dots represent single clones; bars indicate mean ± SD. (C) Local deposition of H3K9me3 and HP1α is unaffected by SetDB1-HMM mutation, but their spreading to distal regions is disrupted. H3K9me3 ChIP-qPCR (left) and HP1α ChIP-qPCR (right) were performed after 7 days of SetDB1 (top) or HP1α (bottom) recruitment. Dots represent independent biological replicates; bars indicate mean ± SD. See for amplicon locations. (D) Defective SetDB1 histone mimic methylation impairs heterochromatin maintenance upon SetDB1 (purple) and HP1α (blue) recruitment. Dox was removed after 14 days of recruitment, and GFP signal was measured by flow cytometry at different time points after Dox removal. The histograms (left) show results from representative single clones. Bar graphs (right) show the quantitative results from different clones. Dots represent single clones; bars indicate mean ± SD. Numbers indicate mean values. Details and color scheme for histograms are the same as in panel B.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Mutagenesis, Stable Transfection, Expressing, Flow Cytometry, Clone Assay, Control, Amplification, Methylation

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: WT or HMm SetDB1 was recruited to the reporter, or HP1α was recruited to the reporter in SetDB1-KO cells rescued with either WT or HMm SetDB1. The expression of the hygromycin resistance gene located 5.5 kb upstream of the tethering site was detected by RT-qPCR. GAPDH was used as an internal reference, and the repression fold was calculated as the fold difference in hygromycin expression in the absence or presence of Dox. The amplicon used for qPCR analysis is shown as a red bar.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Expressing, Quantitative RT-PCR, Amplification

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) Genome-wide H3K9me3 distribution changes in SetDB1-HMM mutant. Circos plot showing H3K9me3 signal differences between cells stably expressing SetDB1-HMm or SetDB1-WT in a SetDB1-KO background. Outer gray tiles represent chromosomes. In the middle circle, black lines indicate 1Mb genomic windows where H3K9me3 ChIP/Input signal >1.5 in the WT rescue strain (defined as heterochromatic region, “het”). Green regions show no significant H3K9me3 enrichment (defined as euchromatin region, “eu”). The inner circle shows log2-transformed H3K9me3 signal changes (positive: orange, negative: blue) in SetDB1-HMm versus WT rescue strain. Data represent the average of two biological replicates. All subsequent ChIP-Seq and RNA-Seq data are from these two rescue strains. (B) The effect of SetDB1-HMm on H3K9 trimethylation varies across genomic regions. Scatterplot shows H3K9me3 ChIP/Input signals in 1Mb genomic windows (gw) from SetDB1-HMm versus WT rescue strain. Data represent the average of two biological replicates. Black dashed lines indicate a 1.5-fold difference. Gray dashed lines separate heterochromatic (H3K9me3 ChIP/Input >1.5) and euchromatic (H3K9me3 ChIP/Input <1.5) windows for both WT and HMm strains. Genomic windows from different chromosomes and with varying fold changes are depicted in different colors. (C) Euchromatin regions with low but detectable H3K9me3 signal gain H3K9me3 in SetDB1-HMM mutant. UCSC browser snapshot shows a representative euchromatin region with elevated H3K9me3. Tracks show overlaid ChIP (colored) and Input (gray) signals from two replicates. Numbers on the right show the normalized ChIP/Input signal for the manually selected genomic interval indicated by the black bar. (D) SetDB1-HMm leads to significant loss of H3K9me3 on ChrX. Left: Dot plot showing the distribution of H3K9me3 ChIP signal in 1Mb genomic windows from ChrX in both WT and SetDB1-HMm strains. Data represent the average of two biological replicates. The black line indicates the median. p<0.0001 is denoted as ****. Right: UCSC browser snapshot of a representative locus on ChrX. Tracks show overlaid ChIP (colored) and Input (gray) signals for two replicates. Numbers on the right show the normalized ChIP/Input signal for the manually selected genomic interval indicated by the black bar. (E) SetDB1-HMm leads to H3K9me3 loss over KRAB-Znf genes. Dot plot shows H3K9me3 signals on individual KRAB-Znf genes in SetDB1-WT and SetDB1-HMm strains. KRAB-Znf genes on Chr19 and on other chromosomes are plotted separately. Data represent the average of two biological replicates. The black line indicates the median. p<0.0001 is denoted as ****, p<0.05 is denoted as *. (F) H3K9me3 spreading on KRAB-Znf genes is impaired in SetDB1-HMM mutant. Left: Metaplot of H3K9me3 distributions over gene bodies of all KRAB-Znf genes in SetDB1-WT (blue) and HMm (orange) strains. Data is average of two biological replicates. Right: UCSC browser snapshot of a representative KRAB-Znf cluster on Chr19. Tracks show overlaid ChIP (colored) and Input (gray) signals for two replicates.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Genome Wide, Mutagenesis, Stable Transfection, Expressing, Transformation Assay, ChIP-sequencing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: H3K9me3 ChIP-seq was performed in SetDB1-KO cells in which SetDB1 loss was rescued by stably expressing either HMm or WT SetDB1. Dot plots show the fold change in H3K9me3 in SetDB1-HMm rescue strain compared to WT rescue strain in 1Mb genomic windows (gw) along the chromosomes. Gray dashed lines indicate a 1.5-fold (1.5x) change. Windows with >1.5x increase in H3K9me3 signal are shown in yellow.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: ChIP-sequencing, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) Violin plots show the proportion of gene (left) and protein-coding gene (right) annotations in the sequence of 1Mb genomic windows. Windows are categorized based on whether the H3K9me3 signal increases by ≥1.5x (yellow) or <1.5x (grey) in SetDB1-HMm rescue strain compared to WT rescue strain. The solid line indicates the median, and the dashed lines represent quantiles. p-value < 0.0001 is indicated by ****. (B) Gene type distribution in regions showing ≥1.5x elevated H3K9me3 signal in SetDB1-HMM mutant (left) and genome-wide (right). (C) Most genes in regions with ≥1.5x elevated H3K9me3 signal in SetDB1-HMM mutant show no or low expression. A dot plot (left) shows log-transformed RPKM values (from RNA-Seq of the WT rescue strain) for genes located in 1Mb windows with ≥1.5x or <1.5x increase in H3K9me3 signal in SetDB1-HMm rescue strain compared to WT rescue strain. The right panel shows the proportions of genes with different expression levels in both groups. Numbers indicate the percentage of genes within each group.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Sequencing, Mutagenesis, Genome Wide, Expressing, Transformation Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: H3K9me3 signal decreased significantly more on ChrX and Chr19 compared to other chromosomes in SetDB1-HMm rescue strain compared to WT rescue strain. Dot plots (bottom) show fold changes of H3K9me3 (HMm/WT) in 1Mb genomic windows. The percentage of windows with ≥1.5x, 1.3-1.5x, or <1.3x loss of H3K9me3 in the mutant was calculated for each group. p-value < 0.0001 is indicated by ****.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: A dot plot shows H3K9me3 enrichment in 1Mb genomic windows across different chromosomes in SetDB1-WT rescue strain. Numbers indicate the percentage of heterochromatic windows (H3K9me3 ChIP/Input >1.5) for each chromosome.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques:

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) H3K9me3 loss correlates with increased gene expression on ChrX. RNA-seq data were analyzed by DESeq2 using 3 replicates for WT and 2 replicates for SetDB1-HMM mutant. ChrX genes located in 1Mb heterochromatic windows with ≥1.5x loss of H3K9me3 in the mutant are shown in blue; all other genes are shown in grey. ChrX genes with significantly increased expression in the mutant (FDR < 0.05) are listed. (B) Heterochromatic KRAB-Znf genes that show ≥1.5x loss of H3K9me3 exhibit mildly increased gene expression in SetDB1-HMm rescue strain. KRAB-Znf genes with ≥1.5x loss of H3K9me3 in the mutant are shown in orange (FDR ≥ 0.05) or red (FDR < 0.05), while all other genes are shown in grey.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Expressing, RNA Sequencing Assay, Mutagenesis

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) KRAB-Znf genes on Chr19 reside in heterochromatin. Violin plots show the percentage of KRAB-Znf gene annotations within 1Mb windows on Chr19. Windows are categorized based on whether they are heterochromatic (H3K9me3 ChIP/Input ≥1.5 in WT rescue strain) or euchromatic (H3K9me3 ChIP/Input <1.5 in WT rescue strain). p-value < 0.0001 is indicated by ****. (B) Regions on Chr19 that lose H3K9me3 signal in SetDB1-HMm strain are enriched in KRAB-Znf genes. Violin plots show the percentage of KRAB-Znf gene annotations within 1Mb windows on Chr19. Windows are categorized based on whether H3K9me3 signal loss in SetDB1-HMm rescue strain was ≥1.3x or <1.3x. p-value < 0.01 is indicated by **.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques:

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: HP1 dimers were shown to stabilize condensed heterochromatin by binding H3K9me3 marks on adjacent nucleosomes. SetDB1 auto-methylates its HMMs creating sites for HP1 binding. In the indirect read-write model, SetDB1 is recruited to chromatin by HP1. In regions with lower density of the H3K9me3 mark, such as newly replicated chromatin (left) and the border region of heterochromatin (right), some HP1 dimers are not binding two histone tails, allowing one CD to bind and recruit SetDB1. SetDB1 methylates adjacent histone tails, maintaining heterochromatin integrity and enabling its spreading into adjacent regions. Accumulation of H3K9me3 displaces SetDB1 from chromatin as the H3K9me3 mark and SetDB1 compete for HP1 binding. Release of SetDB1 enables its redistribution to new, partially methylated regions.

Article Snippet: EGFP-SetDB1 was expressed and purified from HEK293T cells using a modified cell lysis buffer (20mM Tris-Cl pH7.5, 150mM NaCl, 1% DDM, cOmplete EDTA-free protease inhibitor cocktail) and GFP-Trap magnetic agarose beads (Proteintech, gtma).

Techniques: Binding Assay, Methylation

Journal: Journal of Virology

Article Title: Epigenetic Silencing of Recombinant Adeno-associated Virus Genomes by NP220 and the HUSH Complex

doi: 10.1128/jvi.02039-21

Figure Lengend Snippet: sgRNA sequences used in this study

Article Snippet: SETDB1 (catalog no. 11231-1-AP) and MPP8 (catalog no. 16796-1-AP) were purchased from Proteintech.

Techniques: Sequencing

Journal: bioRxiv

Article Title: N 6 -methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics

doi: 10.1101/2025.01.09.632176

Figure Lengend Snippet: a-c , m 6 A profile on lncRNAs Kcnq1ot1 ( a ), Malat1 ( b ), and Neat1 ( c ). Data are from ref . MeRIP-seq data is shown in blue and input data in grey. For each panel, gene structure and transcriptional direction are shown below. Black boxes highlight METTL3 dependent m 6 A modification peaks. d-f , Expression levels for lncRNAs Malat1 ( d ), Neat1 ( e ), and Kcnq1ot1 ( f ) from ChrRNA-seq analysis. Cell lines are indicated at the bottom. Samples are from METTL3-FKBP12 F36V and transgene complementation experiments. Control samples are shown in grey, whereas the dTAG-13 treated (26 hours) samples are shown in black. h-i , As in ( d-f ), but for samples YTHDC1-FKBP12 F36V (left), ZCCHC8-FKBP12 F36V (right), and ZFC3H1-FKBP12 F36V (right). Note that two replicates are shown for each YTHDC1 clone.

Article Snippet: Antibodies used in this study are: METTL3 (Abcam, ab195352), METTL14 (Sigma-Aldrich, HPA038002), RBM15 (Proteintech, 10587-1-AP), YTHDC1 (Sigma-Aldrich, HPA036462), TBP (Abcam, ab51841), SETDB1 (Proteintech, 11231-1-AP), ZCCHC8 (Proteintech, 23374-1-AP), ZFC3H1 (Sigma-Aldrich, HPA007151), and KAP1 (Abcam, ab10484).

Techniques: Modification, Expressing, Control

Journal: bioRxiv

Article Title: N 6 -methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics

doi: 10.1101/2025.01.09.632176

Figure Lengend Snippet: a, Schematic showing the experimental design for SLAM-seq. Cells used in this analysis are in the Xist-BglG expressing lines with -FKBP12 F36V tags on METTL3, YTHDC1, or ZCCHC8. b , Best fit curves indicating Xist degradation determined from SLAM-seq T-to-C conversions. Two independent experiments are shown (top and bottom panels). Black and red represent untreated and dTAG-13 treated samples respectively. c , Dot plot showing the difference of Xist half-life with and without dTAG-13 treatment from (b). Replicates 1 and 2 are shown in blue and orange respectively.

Article Snippet: Antibodies used in this study are: METTL3 (Abcam, ab195352), METTL14 (Sigma-Aldrich, HPA038002), RBM15 (Proteintech, 10587-1-AP), YTHDC1 (Sigma-Aldrich, HPA036462), TBP (Abcam, ab51841), SETDB1 (Proteintech, 11231-1-AP), ZCCHC8 (Proteintech, 23374-1-AP), ZFC3H1 (Sigma-Aldrich, HPA007151), and KAP1 (Abcam, ab10484).

Techniques: Expressing

Journal: bioRxiv

Article Title: N 6 -methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics

doi: 10.1101/2025.01.09.632176

Figure Lengend Snippet: a , Strategy showing in-frame insertion of FKBP12 F36V into ZCCHC8 (top). Western blot (bottom) shows the protein level of ZCCHC8-FKBP12 F36V after 0 or 2 hours dTAG-13 treatment. TBP loading control. The red and black arrows indicate the protein size for ZCCHC8-FKBP12 F36V and untagged ZCCHC8. b , As in (a), but for ZFC3H1. METTL3 loading control. c , Boxplot showing the allelic ratio of X-linked genes from ChrRNA-seq analysis for ZCCHC8 dTAG degron samples. The experimental design is as described in . The red dashed line indicates allelic ratio at 0.5. Samples (two independent clones, Z4F and Z12H) and conditions are indicated above and below respectively. d , As in (c), but for ZFC3H1. Note that only the proximal 138 MB of X chromosome in 11A clone is informative for allelic-speific analysis. e, Barplot showing the expression level of Xist from ChrRNA-seq analysis for samples and conditions described in (c). f, As in (e), but for ZFC3H1 clones described in (d). g, Schematic depicting alternative models for m 6 A-mediated regulation of Xist RNA turnover as discussed in the main text. m 6 A sites are indicated as lollipops with open triangle.

Article Snippet: Antibodies used in this study are: METTL3 (Abcam, ab195352), METTL14 (Sigma-Aldrich, HPA038002), RBM15 (Proteintech, 10587-1-AP), YTHDC1 (Sigma-Aldrich, HPA036462), TBP (Abcam, ab51841), SETDB1 (Proteintech, 11231-1-AP), ZCCHC8 (Proteintech, 23374-1-AP), ZFC3H1 (Sigma-Aldrich, HPA007151), and KAP1 (Abcam, ab10484).

Techniques: Western Blot, Control, Clone Assay, Expressing

Journal: bioRxiv

Article Title: N 6 -methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics

doi: 10.1101/2025.01.09.632176

Figure Lengend Snippet: a , Schematic showing the experimental procedure for control or 26 hours dTAG-13 treated samples for ChrRNA-seq analysis. b , Aggregated profile (top) and heatmap (bottom) showing the expression of expressed genes (blue) and their promoter upstream transcripts (PROMPTs) (red). The negative value shown in red in this plot indicates antisense transcription. Plots show data for transcription start site ± 2.5 kb. c , Genome browser screenshot of ChrRNA-seq of the Xist gene from ZCCHC8 degron samples with Dox or dTAG-13 + Dox treatment. d , PCA using allelic ratio of X-linked genes (n=237) for samples of acute depletion of ZCCHC8 in or YTHDC1 in , along with time-course WT (iXist-ChrX 129 , A11B2 clone) cells, cells with SPEN knockout or SPOC mutant, and cells with Xist B/C-repeat deletion (data from ref ). Note that X-linked gene silencing is defective in SPEN knockout, SPOC mutant, and Xist B/C-repeat deletion, thus datapoints representing those samples lie away from the predicted silencing trajectory.

Article Snippet: Antibodies used in this study are: METTL3 (Abcam, ab195352), METTL14 (Sigma-Aldrich, HPA038002), RBM15 (Proteintech, 10587-1-AP), YTHDC1 (Sigma-Aldrich, HPA036462), TBP (Abcam, ab51841), SETDB1 (Proteintech, 11231-1-AP), ZCCHC8 (Proteintech, 23374-1-AP), ZFC3H1 (Sigma-Aldrich, HPA007151), and KAP1 (Abcam, ab10484).

Techniques: Control, Expressing, Knock-Out, Mutagenesis

Journal: Journal of ovarian research

Article Title: SETDB1 promotes progression through upregulation of SF3B4 expression and regulates the immunity in ovarian cancer.

doi: 10.1186/s13048-024-01358-8

Figure Lengend Snippet: Fig. 2 Overexpression and knockdown efficiency of SETDB1. A The efficiency of SF3B4 mRNA overexpression and knockdown was detected by qRT-PCR. B Up- and down-regulation efficiency of SETDB1 protein was confirmed by WB

Article Snippet: WB was performed with specific antibodies against SETDB1 (1:1000, Proteintech, 11,231–1-AP), SF3B4 (1:8000, Proteintech, 10,482–1-AP), and β-actin (1:8000, SigmaAldrich, SAB3500350).

Techniques: Over Expression, Knockdown, Quantitative RT-PCR

Journal: Journal of ovarian research

Article Title: SETDB1 promotes progression through upregulation of SF3B4 expression and regulates the immunity in ovarian cancer.

doi: 10.1186/s13048-024-01358-8

Figure Lengend Snippet: Fig. 5 SETDB1 promotes the expression of SF3B4 in ovarian cancer. A Cistrome DB data showed the binding peak of SETDB1 protein in the SF3B4 promoter region. B Visualization analysis of Cistrome project 2124 data by IGV showed the SETDB1 binding site in SF3B4 promoter. C TCGA-OV database showed the positive correlation between SETDB1 and SF3B4 mRNA expression in ovarian cancer. D, E qRT-PCR and WB showed the effect of SETDB1 knockdown on SF3B4 mRNA and protein expression in ovarian cancer. F Luciferase reporter assays showed the effect of SETDB1 on SF3B4 promoter in HEY cells

Article Snippet: WB was performed with specific antibodies against SETDB1 (1:1000, Proteintech, 11,231–1-AP), SF3B4 (1:8000, Proteintech, 10,482–1-AP), and β-actin (1:8000, SigmaAldrich, SAB3500350).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Knockdown, Luciferase

Journal: Journal of ovarian research

Article Title: SETDB1 promotes progression through upregulation of SF3B4 expression and regulates the immunity in ovarian cancer.

doi: 10.1186/s13048-024-01358-8

Figure Lengend Snippet: Fig. 6 Knockdown of SF3B4 weakens the biological effects of SETDB1 overexpression in ovarian cancer cells. A MTT assay detected the effect of SETDB1 and SF3B4 different expression on proliferation in HEY cells. B, C Transwell assays showed the effect on migration and invasion ability of SF3B4 deficiency on SETDB1 stable-overexpressed HEY cells

Article Snippet: WB was performed with specific antibodies against SETDB1 (1:1000, Proteintech, 11,231–1-AP), SF3B4 (1:8000, Proteintech, 10,482–1-AP), and β-actin (1:8000, SigmaAldrich, SAB3500350).

Techniques: Knockdown, Over Expression, MTT Assay, Expressing, Migration

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Frequencies of SLC38 family genetic alterations in integrated ESCC cohort in our data

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques:

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: CNA and clinical association of SLC38A3 in ESCC integrated cohort and TCGA cohort. (A) CNA loss frequency of SLC38A3 in ESCC integrated cohort and TCGA cohort; (B) Scatter plot showed relative copy number variations of SLC38A3 in ESCC integrated cohort and TCGA cohort; (C) Correlation between SLC38A3 CNA and mRNA level in TCGA cohort (r=0.2909, P=0.004, Pearson Correlation Coefficient test); (D,E) Kaplan-Meier survival analysis of ESCC integrated cohort stratified by SLC38A3 CNA loss (n=314; P=0.011 for OS and P=0.014 for 5-year survival, log-rank test). CNA, copy number alteration; SLC38, solute carrier family 38; ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas; OS, overall survival.

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques:

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Association of SLC38A3 CNV with clinicopathological features in integrated ESCC cohort

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques:

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Clinical implication of SLC38A3 in ESCC tumor samples. (A) Representative IHC photos of SLC38A3 expression in ESCC sample and normal tissue sample. Scale bar: 200 μm (upper) and 50 μm (bottom); (B) Quantitative IHC scores of SLC38A3 in normal tissue samples (n=75) and ESCC samples (n=105, Student’s t -test); (C) Quantitative IHC scores of SLC38A3 in 75 paired normal tissue samples and ESCC samples (paired Student’s t -test); (D,E) Kaplan-Meier survival analysis of ESCC tissue microarray stratified by SLC38A3 CNA loss (n=104; P=0.002 for OS and P=0.004 for 5-year survival, log-rank test). ***, P<0.001. SLC38s, solute carrier family 38; ESCC, esophageal squamous cell carcinoma; IHC, immunohistochemistry; OS, overall survival.

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Expressing, Microarray, Immunohistochemistry

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Association of SLC38A3 expression with clinicopathological features in ESCC TMA cohort

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Expressing

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Multivariate Cox regression analysis of risk factors associated with OS and 5-year survival

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Expressing

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Loss of SLC38A3 promotes cell proliferation, migration and invasion of ESCC. (A) Western blotting analysis of SLC38A3 in nine ESCC cell lines and two immortalized normal esophageal cell lines NE2 and NE3; (B) Western blotting analysis of SLC38A3 expression after introducing small interfering RNA (siRNA) of SLC38A3 in KYSE410 and KYSE510. Cell lysates were collected 48 h after transfection; (C) Cell proliferation assays were performed after introducing siRNA of SLC38A3 in KYSE410 and KYSE510, and cell viability was quantified by MTS assay. Data represent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\large \overline{\textit{x}}{\text{±}}{\textit{s}}_\overline{\textit{x}}$\end{document} , n=3 independent experiments. Unpaired t test was performed to calculate P values; (D) Colony formation assays with siCtrl, siSLC38A3_1, siSLC38A3_3 transfected cells for 10 d. Data represent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\large \overline{\textit{x}}{\text{±}}{\textit{s}}_\overline{\textit{x}}$\end{document} , n=3 independent experiments. Unpaired t test was performed to calculate P values; (E) Transwell-based migration and invasion assay with siCtrl, siSLC38A3_1, siSLC38A3_3 transfected cells for 12 h. Scale bars: 50 µm. Data represent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\large \overline{\textit{x}}{\text{±}}{\textit{s}}_\overline{\textit{x}}$\end{document} , n=3 independent experiments. Unpaired t test was performed to calculate P values; (F) Wound healing assay of siCtrl, siSLC38A3_1, siSLC38A3_3 transfected cells for 24 h. Representative images were taken with phase contrast microscopy. Scale bars: 50 µm; (G) Representative images of siCtrl, siSLC38A3_1 transfected cells with or without TGF-β (10 ng/mL) for 48 h. Scale bars: 50 µm. **, P<0.01; ***, P<0.001; ****, P<0.0001 for three independent experiments analyzed by Student’s t test. SLC38s, solute carrier family 38; ESCC, esophageal squamous cell carcinoma; TGF-β, transforming growth factor beta.

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Migration, Western Blot, Expressing, Small Interfering RNA, Transfection, MTS Assay, Invasion Assay, Wound Healing Assay, Microscopy

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: SLC38A3 suppresses transcription of SNAIL by interacting with SETDB1. (A) Western blotting analyses of expression of indicated proteins in siCtrl; (B) Immunofluorescence images of SLC38A3, SETDB1 in KYSE510 cells. Scale bars: 5 µm; (C) Western blotting analyses of immunoprecipitation (IP) products and whole cell lysates derived from KYSE410; (D) Western blotting analyses of expression of total SETDB1 in siCtrl, siSLC38A3_1, siSLC38A3_3 transfected cells derived from KYSE510; (E) Western blotting analyses of expression of indicated proteins in siCtrl, siSLC38A3, siSETDB1 and siSLC38A3+siSETDB1 transfected cells derived from KYSE410; (F) Quantitative RT-PCR analyses of SNAIL levels in siCtrl, siSLC38A3, siSETDB1 and siSLC38A3+siSETDB1 transfected cells derived from KYSE410. SLC38s, solute carrier family 38; IgG, immunoglobulin G; RT-PCR, real-time polymerase chain reaction.

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Western Blot, Expressing, Immunofluorescence, Immunoprecipitation, Derivative Assay, Transfection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: List of potential interactors of SLC38A3*

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques:

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: SLC38A3 expression level correlated with several target therapeutic drugs. (A) Volcano plot displaying association between SLC38A3-loss and 251 pharmacologic inhibitors from GDSC study. X axis, Pearson correlation coefficient r; Y axis, significance −log 10 P (P<0.05, r>0.4 or r<−0.4); (B) Heatmap showed IC 50 of SLC38A3 significantly correlated drugs in different ESCC cell lines. The ln (IC 50 ) data were used for z-score calculation. SLC38s, solute carrier family 38; GDSC, Genomics of Drug Sensitivity in Cancer database; IC 50 , half inhibitory concentration; ESCC, esophageal squamous cell carcinoma.

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Expressing, Concentration Assay

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Sensitivities of significant SLC38A3 mRNA level associated drugs in ESCC cell lines

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques:

Journal: Chinese Journal of Cancer Research

Article Title: Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

doi: 10.21147/j.issn.1000-9604.2020.05.01

Figure Lengend Snippet: Sensitivities of significant SLC38A3 LOH associated drugs in ESCC cell lines

Article Snippet: The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: