Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation decreases methyltransferase activity and intrinsic protein stability of SETD2 catalytic core in vitro . A , upper panel : schematic representation of the SETD2 domains. The SETD2 L1609P mutation is located in the SET domain within the SETD2 catalytic core (composed of the AWS, SET, and post-SET domains). Lower left panel : Structural representation of the SETD2 active site (PDB entry: 5JJY ) with a zoomed-in view of the substrate (H3K36M peptide) and cofactor (SAH) binding sites. Lower right panel : Sequence alignment of residues 1603 to 1619 of the SET domain of human SETD2 with the equivalent sequences of human G9A, EZH2, NSD1, NSD2, SETD8, MLL1, MLL2, SETD8, ASH1 (sequence retrieved from the UniProt database). Conserved residues are highlighted in blue . The secondary structure of the SETD2 residues (deduced from PDB entry: 5JJY ) is shown above the alignment. The SETD2 residue L1609 and the equivalent residues in the other SET domain-containing enzymes are highlighted in orange . B , in vitro methylation of recombinant histone H3, core histones (purified from HEK293T SETD2-KO cells) or recombinant nucleosomes. SETD2-dependent H3K36me3 methylation was detected using an anti-H3K36me3 antibody. Ponceau Red staining of histones is shown. The purified catalytic core of SETD2 WT and SETD2 L1609P mutant used in the assays were detected using an anti-6xHis-tag antibody. C , SETD2 mono-methylation, dimethylation, or trimethylation activities were determined by UFLC assays using H3K36 fluorescent peptides as previously described ( , ). Bar graphs and error bars represent the mean and SD of three independent experiments. D , automethylation of SETD2 and methylation of α-tubulin detected by autoradiography using 3 H-SAM. Coomassie Blue staining was used as loading control. E , determination of the intrinsic protein stability of SETD2 WT or SETD2 L1609P by thermal shift assay (TSA). Left panel : T m values were determined by the minimum of the first derivative of the fluorescence emission as a function of temperature (dFluo/dT). Right panel : Bar graphs and error bars represent the mean and SD of nine experiments. SETD2, SET-domain containing protein 2; UFLC, ultrafast liquid chromatography.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Activity Assay, In Vitro, Binding Assay, Sequencing, Residue, Methylation, Recombinant, Purification, Staining, Autoradiography, Control, Thermal Shift Assay, Fluorescence, Liquid Chromatography

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation results in low levels of the H3K36me3 mark and in low expression of SETD2 in CRISPR/Cas9-engineered HEK293T cells and in transfected HEK293T-SETD2 KO cells . A , endogenous H3K36me3 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : the H3K36me3 mark was detected by immunofluorescence using an anti-H3K36me3 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with 10 μm scale bars. Right panel : Histones from CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant were extracted and H3K36me3 levels were determined by Western blotting using a an anti-H3K36me3 antibody. Ponceau Red staining of extracted histones is shown. B , endogenous SETD2 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : Cells were fixed and SETD2 was detected using an anti-SETD2 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with scale bars of 10 μm. Right panel : SETD2 was detected in cell extracts by Western blot using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. C , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 L1609P were transfected with GFP-SETD2 WT or GFP-SETD2 L1609P plasmids. Nontransfected CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were used as controls. Ectopic GFP-SETD2 expression and H3K36me3 mark levels were detected by Western blot using anti-GFP or anti-H3K36me3 antibodies, respectively. Ponceau Red staining of cellular histones or extracts on membranes are shown. D , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were treated with MG132 or DMSO. Endogenous SETD2 WT and SETD2 L1609P expression levels were detected by Western blotting using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Expressing, CRISPR, Transfection, Immunofluorescence, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Overall structure of the ternary complex of SETD2 L1609P mutant bound to H3K36M peptide and SAM cofactor . A , left panel : cartoon representation of SETD2 WT (PDB: 5JJY ) ( cyan ) bound to H3K36M peptide ( orange ) and the SAH cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 L1609 and H3M36 residues are represented by yellow and orange sticks , respectively. The close-up view shows the region around residue L1609 with the H3K36M peptide (residues 29–42, orange ) and the SAH cofactor ( black sticks ). Zinc atoms are shown in gray . Right panel : cartoon representation of the SETD2 L1609P mutant (PDB: 8RZU ) ( salmon ) bound to the H3K36M peptide ( green ) and the SAM cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 P1609 and H3M36 residues are shown as yellow and green sticks , respectively. The close-up view shows the region around the residue P1609 with the H3K36M peptide (residues 29–39, green ) and the SAM cofactor ( black sticks ). B , left panel : cartoon representation of the characteristic triangular shape of the SET domain formed by 3 β-sheets (β1-β2; β3-β8-β7; β4-β6-β5 strands) of SETD2 WT in complex with the H3K36M peptide (residues 29–42 in orange) (PDB: 5JJY ). The β-sheet composed of β4-β6-β5 strands is boxed and the SETD2 L1609 residue is shown in yellow . Right panel : cartoon representation of the triangular β-sheet structure of the SET domain of the SETD2 L1609P mutant ( salmon ) in complex with the H3K36M peptide (residues 29–39, green ) (PDB: 8RZU ). The β5-strand in SETD2 WT adopts a loop conformation in the structure of the SETD2 L1609P mutant ( boxed ). The P1609 residue in mutant SETD2 is shown in yellow . SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Effects of the SETD2 L1609P mutation on the conformations of neighboring residues of SETD2 and the H3K36M peptide. A , the left panel shows a cartoon overlay of the β5-β6 hairpin of SETD2 WT (PDB: 5JJY ) ( cyan ) and SETD2 L1609P mutant ( salmon ) structures. The H3K36M peptide is shown in orange and green for SETD2 WT and SETD2 L1609P, respectively. The side chains of residues L1609 and P1609 residues are shown as sticks ( yellow CPK). The middle panel shows a close-up view of the hairpin residues (1609–1613) of SETD2 WT ( cyan ) and SETD2 L1609P ( salmon ). The side chains are shown in CPK sticks . The right panel shows the β5-β6 hairpin residues of SETD2 WT ( top ) and SETD2 L1609P ( bottom ) in sticks . Dashes represent the distance between Cα of residues K1610 and E1613 residues. B , conformational remodeling of residues K1610 and K1639 of SETD2 and residue K37 of H3 induced by the L1609P mutation. Left panel shows residues SETD2 L1609 ( yellow ), K1610 (cyan), K1639 ( cyan ), and H3K37 ( orange ) in spheres and sticks in the SETD2 WT structure (PDB: 5JJY ). Middle panel shows residues SETD2 P1609 ( yellow ), K1610 ( salmon ), K1639 ( salmon ), and H3K37 ( green ) in spheres and sticks in the SETD2 L1609P structure. The right panel shows residues P1609 ( yellow ) and K1610 ( salmon ) from the SETD2 L1609P structure and residues K1639 ( cyan ) and H3K37 ( orange ) from the SETD2 WT structure. Steric clashes between side chains are shown in boxes . The orientations are the same in all three panels and were obtained by superimposing the SETD2 WT and L1609P main chains. C , surface representation of the SETD2 substrate-binding region. H3K36M peptides are shown as sticks. The left panel shows the SETD2 WT structure (PDB: 5JJY ) in light cyan . The SETD2 L1609 residue is shown in yellow . The SETD2 K1610 and K1639 residues are shown in blue . H3K36M peptide residues diffracting in both WT and L1609P structures (residues A29–H39) are shown in green . H3K36M peptide residues observed only in the SETD2 WT structure (residues R40-R42) are shown in transparent orange . The right panel shows the SETD2 L1609P structure in light pink . The SETD2 P1609 residue is shown in yellow . The K1610 and K1639 residues are shown in purple . H3K36M peptide residues observed in the SETD2 L1609P structure (A29–H39) are shown in green . SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Details of H3K36M peptide recognition by SETD2 L1609P mutant . A , the left panel shows a clipped surface representation of the SETD2 WT-H3K36M peptide complex (PDB: 5JJY ). Peptide residues (residues A29–R42) are represented by sticks . The right panel shows a clipped surface representation of the SETD2 L1609P-H3K36M peptide complex. Peptide residues (A29–H39) are represented by sticks . The structures of the SETD2-H3K36M peptide complexes are shown in the same orientation after superimposition of the main chains. B , upper panel : Structural alignment of H3K36M peptides (residues A29–H39) in SETD2 WT (PDB: 5JJY ) ( orange ) and SETD2 L1609P ( green ) structures. Lower panel : Differences between SETD2-H3K36M peptide interactions in SETD2 WT and SETD2 L1609P complexes. Residue interactions across the binding interface of SETD2 WT or SETD2 L1609P mutant with H3K36M peptide were determined using LIGPLOT . Residues are represented by sticks . Residues involved in SETD2-H3K36M peptide interactions (nonbonded and hydrogen bonds) are represented by sticks and spheres . Dashes represent hydrogen bond. The lower left panel shows the SETD2 WT ( cyan )-H3M36 ( orange ) interacting residues that are specific for the SETD2 WT complex and not present in the SETD2 L1609P complex. These interactions are listed in a table ( bottom left ). The lower right panel shows SETD2 L1609P ( salmon )-H3K36M ( green ) peptide interacting residues that are specific for the SETD2 L1609P complex and not present in the SETD2 WT complex. These interactions are listed in a table ( bottom right ). SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) The growth response of wild-type S. pombe , set2Δ, wee1-50, paf1Δ, set2Δ wee1-50, set2Δ paf1Δ, and set2Δ wee1-50 paf1Δ was compared by spotting 5-fold serial dilutions of each strain onto YE6S medium. Plates were the incubated for an appropriate time at either 25, 32 or 35.5 °C. ( B-C ) SETD2 WT and SETD2 CRISPR KO U2OS cells after 72 h exposure to different concentrations of AZD1775 (75, 150, 300 and 600 nM). (D-E) Viability assay to examine the impact of other PAF1 complex factors (including CDC73 and CTR9) silencing on AZD1775-sensitivity. Survival fraction of non-targeting control, CDC73, and CTR9 siRNA treated SETD2 WT and SETD2 CRISPR KO cells after 72 h exposure to different concentrations of AZD1775 (75, 150, 300 and 600 nM). Data points and bars represent the mean and SEM of ≥ three independent experiments; * P < 0.05; ** P < 0.01; and *** P < 0.001. (F) Viability assay to examine the impact of PAF1 silencing on HU or gemcitabine. Survival fraction of non-targeting control and PAF1 siRNA treated SETD2 WT and SETD2 CRISPR KO U2OS cells after 72 h exposure to different concentrations of HU (150, 300, 600 and 1200 uM) or gemcitabine (12.5, 25, 50 or 100 nM). Data points and bars represent the mean and SEM of ≥ three independent experiments; * P < 0.05; ** P < 0.01; and *** P < 0.001. (G) Viability assay to examine the sensitivity to HU in gene deletions of PAF1C components in S. pombe .

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Incubation, CRISPR, Viability Assay, Control

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) Validation of PAF1 siRNA knockdown efficiency by Western Blot in SETD2 WT and SETD2 CRISPR KO U2OS cells 72 hours post-siRNA treatment. (B) Viability assay to examine the impact of PAF1 silencing on AZD1775-sensitivity. Survival fraction of non-targeting control and PAF1 siRNA treated (A-B) 786-0 ( SETD2 WT) and A498 (SETD2-mutant) renal cancer cells. C) Cleaved PARP, procaspase 3, cleaved caspase 3 and actin protein expression in SETD2 WT and SETD2 CRISPR KO U2OS cells 48 hours post-treatment with either 200 nM DMSO or 200 nM AZD1775 was assessed using Western Blot. D) Validation of CDC73 and CTR9 siRNA knockdown efficiency by Western Blot in SETD2 WT and SETD2 CRISPR KO U2OS cells 72 hours post-siRNA treatment.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Biomarker Discovery, Knockdown, Western Blot, CRISPR, Viability Assay, Control, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) 48 hours following DMSO or AZD1775-treatment (300 nM), control or siPAF1 treated SETD2 KO U2OS cells were pulse-labelled with BrdU for 30 min and collected for cell cycle analysis. The data shown are from a single representative experiment out of three repeats. Numbers are the relative percentage of cell cycle stage. (B) Quantification of data shown in (A). (C) SETD2 CRISPR KO U2OS were treated with control or siPAF1 siRNA and exposed to 250 nM of AZD1775 for 48 hours. One representative experiment showing yH2AX vs DNA (left panel) and EdU vs DNA (right panel). The numbers indicate the percentages of cells in the high yH2AX-gated population (shown in black box). These cells correspond with the non-replicating S-phase cells as shown in the EdU plots (blue population). (D) Quantification of the yH2AX medium and high populations shown in (C). (E) Western blot analysis of SETD2 CRISPR KO U2OS cells exposed to either non-targeting control siRNA or PAF1 siRNA and/or treated with either DMSO or 300 nM AZD1775. Lamin B was used as a loading control. (F) 48 hr following AZD1775-treatment, control, siCDC73, or siCTR9 treated SETD2 KO U2OS cells were pulse-labelled with BrdU for 30 min and collected for cell cycle analysis.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Control, Cell Cycle Assay, CRISPR, Western Blot

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) SETD2 WT and (B) SETD2 CRISPR KO U2OS were treated with control, siCDC73, siCTR9 or siPAF1 and subsequently synchronized at the G 1 /S transition by a double thymidine block (DTB) and then released by addition of fresh medium. Cells were incubated with BrdU for 30 min and collected for cell cycle analysis 4, 8, and 12 hours after release. (C) Western blot analysis of RRM2 protein levels in SETD2 WT and SETD2 CRISPR KO U2OS cells exposed to either non-targeting control siRNA or PAF1 siRNA and/or treated with either DMSO or 300 nM AZD1775. Tubulin was used as a loading control.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: CRISPR, Control, Blocking Assay, Incubation, Cell Cycle Assay, Western Blot

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) Western blot analysis of p21 protein levels in SETD2 WT and SETD2 CRISPR KO U2OS cells after 48 hours of treatment with either non-targeting control siRNA or PAF1 siRNA. Tubulin was used as a loading control. (B) qRT-PCR analysis of CDKN1A levels normalised to 18S in U2OS cells transfected with the indicated siRNAs (n = 3). (C) Western Blot analysis of p-CDK(S) activity following siPAF1 with and without AZD1775 in SETD2 WT U2OS cells. (D) RNA:DNA hybrid slot blot of genomic DNA ± RNaseH1 treatment from cells treated with siNT, siSETD2, siPAF1 or combined siSETD2 and siPAF1. (E) Western blot analysis of p21 protein levels in SETD2 KO U2OS cells. Immunoblot staining for the V5-epitope confirms RNAseH1 overexpression.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Western Blot, CRISPR, Control, Quantitative RT-PCR, Transfection, Activity Assay, Dot Blot, Staining, Over Expression

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: A) 48 hr following DMSO -or AZD1775-treatment, control, siCDKN1A, siPAF1, or siCDKN1A + siPAF1 treated SETD2 KO U2OS cells were pulse-labelled with BrdU for 30 min and collected for cell cycle analysis. The data shown are from a single representative experiment out of three repeats. Numbers are the relative percentage of cell cycle stage. B) 48 hr following DMSO, AZD1775- and/or UC2288 treatment, SETD2 KO U2OS cells were collected for cell cycle analysis. C) SETD2 WT and SETD2 CRISPR KO U2OS cells after 48h exposure to different concentrations of AZD1775 (0-10 μ M) and UCC2288 (p21 inhibitor).

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Control, Cell Cycle Assay, CRISPR

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) 48 hr following DMSO -or AZD1775-treatment, siPAF1, or siCDKN1A + siPAF1 treated SETD2 KO U2OS cells. The data shown are from a single representative experiment out of three repeats. Numbers are the relative percentage of cell cycle stage. (B) SETD2 CRISPR KO U2OS cells treated with siNT or siPAF1 after 48h exposure to different concentrations of AZD1775 (0-10 μM) and UCC2288 (p21 inhibitor). Survival fraction of non-targeting control, CDC73, and CTR9 (C) siRNA treated TP53 CRISPR KO cells after 72 h exposure to different concentrations of AZD1775 (75, 150, 300 and 600 nM). Data points and bars represent the mean and SEM of ≥ three independent experiments; * P < 0.05; ** P < 0.01; and *** P < 0.001. (D) Western blot analysis of p21 protein levels in SETD2 WT and SETD2 CRISPR KO U2OS cells exposed to either non-targeting control siRNA or p21 siRNA. Tubulin was used as a loading control. (E) Cell viability is further reduced when WEE1 inhibition is combined with p21 knock down in SETD2 WT and SETD2 mutant cancer cell lines. (F) Model where loss of CDC73, CTR9 and PAF1 results in resistance to WEE1i treatment.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: CRISPR, Control, Western Blot, Inhibition, Knockdown, Mutagenesis

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Summary of protein expression losses in ccRCC tumors

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Expressing

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: ( A ) How a phylogenetic tree was constructed. A: ARID1A loss; M: SMARCA2 loss; P: PBRM1 loss, G: SMARCA4 loss; S: SETD2 loss. ( B ) Truncal losses of the markers at each stage, either alone or in combination, were presented. ( C ) Fisher's exact tests were performed to calculate the p values of the associations between the protein marker losses and stages.

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Construct, Marker

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: The survival curves were calculated based on SETD2 staining: positive (1) and negative (0). Associated log-rank p value was indicated. n: number of cases.

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Staining

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Univariate and multivariable analyses of indicated biomarker losses and their associations with overall survival

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Univariate and multivariable analyses of indicated biomarker losses and their associations with recurrence-free survival

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Biomarker Discovery

Journal: PLOS One

Article Title: Identification of novel antiviral host factors by functional gene expression analysis using in vitro HBV infection assay systems

doi: 10.1371/journal.pone.0314581

Figure Lengend Snippet: Primers used in this study. Primers used in custom TaqMan® Array Fast plate.

Article Snippet: 82 , SETD2 , Human , FAM , Hs00383442_m1 , Applied Biosystems.

Techniques:

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 3. HIF1 can directly induce the expression of LBH under hypoxia a: LBH mRNA expression of T98G (left) and GSC4B (right) gradually increased during prolonged treatment under hypoxia as measured by qPCR. (T98G: p <0.0001, GSC4B: p <0.0001, One-Way ANOVA) b: LBH protein expression of T98G and GSC4B was gradually increased during prolonged treatment under hypoxia as measured by western blotting. c: Sequence motif representing the consensus HIF-1 binding motif (JASPAR database). d, e: Luciferase reporter assays showed hypoxia can upregulate the luciferase promoter activities of LBH in T98G (left) and GSC4B (right) cells. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) e: ChIP qPCR showed HIF-1 binding to the promoter of LBH under hypoxia. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) g, h: qPCR (g) and western blot (h) showed the expression of HIF-1 overexpression can upregulate the expression of LBH. (T98G: p <0.0001, GSC4B: p <0.0001, Student’s t-test) All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: Expressing, Western Blot, Sequencing, Binding Assay, Luciferase, ChIP-qPCR, Over Expression

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 5. LBH can activate VEGFA-mediated ERK signalling in hBMECs under hypoxia a, b: The VEGFA mRNA expression and secretion level of U118 and GSC2A cells was upregulated under hypoxia and further upregulated after LBH overexpression, as measured by qPCR (a) and ELISA (b). (qPCR: U118: p <0.0001, GSC2A: p <0.0001; ELISA: U118: p = 0.0019, GSC2A: p = 0.0011, One-Way ANOVA) c, d: The VEGFA mRNA expression and secretion level of T98G and GSC4B cells was upregulated under hypoxia and decreased after LBH knockdown, as measured by qPCR (c) and ELISA (d). (qPCR: T98G: p <0.0001, GSC4B: p = 0.0012; ELISA: T98G: p = 0.0023, GSC4B: p = 0.0029, One-Way ANOVA) e: The VEGFA protein expression of U118 and GSC2A cells was upregulated under hypoxia and further upregulated after LBH overexpression, as shown by western blotting. f: The VEGFA protein expression of T98G and GSC4B cells was upregulated under hypoxia and decreased after LBH knockdown, as shown by western blotting. g: The VEGFR-ERK signalling pathway in vascular endothelial cells following treatment with LBH-silenced T98G and GSC4B conditioned media under normoxia or hypoxia was measured by western blotting. All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 6. Anti-VEGFA treatment can abolish LBH-induced hBMECs proliferation, invasion and angiogenesis under hypoxia a, b: The induction of vascular endothelial cell viability following treatment with LBH-overexpressed U118 (a) and GSC2A (b) conditioned media was reversed following anti-VEGFA treatment, as measured by an MTS assay. (U118: p = 0.0016, GSC2A: p = 0.0011, One-Way ANOVA) c: The proliferation of vascular endothelial cells following treatment with LBH-overexpressed U118 and GSC2A conditioned media was reversed following anti-VEGFA treat- ment, as measured by an EDU incorporation assay. Scale bar = 100 μm. (U118: p = 0.0008, GSC2A: p < 0.0001, One-Way ANOVA) d: Representative transwell assay showing that treatment with LBH-overexpressed U118 and GSC2A conditioned media induced invasion of vascular endothelial cells that was reversed after anti-VEGFA treatment. Scale bar = 100 μm. (U118: p <0.0001, GSC2A: p = 0.0018, One-Way ANOVA) e: Representative tube formation assay showing that treatment with LBH-overexpressed U118 and GSC2A conditioned media induced tubulogenesis of vascular endothelial cells that was reversed after anti-VEGFA treatment. Scale bar = 100 μm. (number of branches: U118: p <0.0001, GSC2A: p < 0.0001, tubule length: U118: p = 0.0012, GSC2A: p = 0.0019, One-Way ANOVA) All data are shown as the mean ± SD (three independent experiments). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: MTS Assay, Transwell Assay, Tube Formation Assay

Journal: EBioMedicine

Article Title: Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

doi: 10.1016/j.ebiom.2019.09.037

Figure Lengend Snippet: Fig. 7. LBH regulates glioma tumorigenesis and angiogenesis in vivo a, c: Representative photographs showing the size of intracranial tumors in the coronal position. LBH overexpression in GSC2A cells increased the intracranial tumor size (a), whereas LBH knockdown in GSC4B cells decreased the intracranial tumor size (c). Scale bar = 10 mm. b, d: LBH-overexpressed GSC2A cells implanted into tumor-bearing mice showed shorter survival times as measured by Kaplan–Meier survival curves (b), compared with longer survival times when LBH-silenced GSC4B cells were implanted into tumor bearing mice (d). For each group, n = 5. e: Representative immunohistochemical staining showing the changes in LBH, VEGFA and CD31 in LBH overexpression and knockdown orthotopic xenograft models. Scale bar = 50 μm. f: Schematic diagram to illustrate that overexpression of LBH promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia.

Article Snippet: Relevant primary antibodies against LBH (1:500, #ab122223), HIF-1 (1:500, #ab1), VEGFA (1:1000, #ab52917), VEGFR2 (1:1000, #ab11939), p-VEGFR2 (1:1000, #ab194806), MEK1/2 (1:1000, #ab178876), p-MEK1/2 (1:1000, #ab96379), ERK1/2 (1:500, #ab17942), p-ERK1/2 (1:500, #ab223500) and βactin (1:2000, #66009–1-Ig, ProteinTech, Chicago, IL, USA) were incubated with the membrane at 4 °C overnight.

Techniques: In Vivo, Over Expression, Knockdown, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: A role for gene expression and mRNA stability in nutritional compensation of the circadian clock

doi: 10.1101/2022.05.09.491261

Figure Lengend Snippet: U2OS cells were transfected with the indicated siRNAs (15 pmol total), incubated for 2 days, and Bmal1-dLuc rhythms were measured for 5 – 6 days following dexamethasone synchronization. Knocked-down cells were assayed in either MEM high glucose (yellow lines) or MEM low glucose (blue lines) medium. Averaged luciferase traces are shown for each glucose concentration without detrending or further data manipulation (N = 6 biological / technical replicates for AllStars negative controls; N = 4 biological / technical replicates for other siRNA knockdowns per nutrient concentration; standard deviation error bars). siRNA knockdown efficiency was measured using RT-qPCR relative to non-transfected control samples (Materials and Methods). ∼90% knockdown was achieved for CPSF6 , and ∼70% knockdown for SETD2 under these experimental conditions (N = 2 biological replicate samples each with N = 3 technical replicate RT-qPCR reactions). Period lengths were calculated and averaged for all replicates ( A ). siRNA knocked-down cells were assayed in either DMEM 30 mM glucose (yellow lines) or DMEM 10 mM glucose (blue lines) medium. One representative luciferase trace is shown (N = 7 – 10 biological / technical replicates per nutrient concentration). Period lengths were calculated and averaged for all replicates and plotted as boxplots ( B ). Average period lengths were: 23.2 ± 1.1 hrs (low glucose control) and 23.5 ± 0.6 hrs (high glucose control); 23.6 ± 1.1 hrs (low glucose SETD2 knockdown) and 25.2 ± 1.8 hrs (high glucose SETD2 knockdown) (* p < 0.05, student’s t-test).

Article Snippet: Although the Opti-MEM medium formulation is not publicly available, one study reported the Opti-MEM glucose concentration as 2.5 g/L or 13.88 mM ( ). siRNAs were obtained from Qiagen: AllStars Negative Control siRNA (Qiagen # 1027280); human SETD2 siRNA (Qiagen # 1027416, FlexTube GeneSolution GS29072, 4x siRNAs used at 3.75 pmol each); human CPSF6 siRNA (Qiagen # 1027416, FlexTube GeneSolution GS11052, 4x siRNAs used at 3.75 pmol each); human CRY2 siRNA (Qiagen # 1027416, FlexTube GeneSolution GS1408, 4x siRNAs used at 3.75 pmol each) ( ).

Techniques: Transfection, Incubation, Luciferase, Concentration Assay, Standard Deviation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

doi: 10.1038/s41467-021-21663-w

Figure Lengend Snippet: a Cartoon illustrating the overlapping segments of SETD2 used for affinity purification along with the known domains. AWS associated with SET, SET Su(var)3–9, Enhancer-of-zeste and Trithorax, SRI Set2-Rpb1 interaction. b Microscopy images showing localization of GFP-SETD2 fragments. The scale bar is 10 µm. The experiment was repeated at least eight times all yielding similar results. c Halo purification was performed from extracts of 293T cells expressing Halo-SETD2C. Input and eluted samples were resolved on a gel followed by silver staining. M—protein marker. The experiment was repeated at least 10 times all yielding similar results. d Table showing the dNSAFs (distributed normalized spectral abundance factor) of the listed proteins. e IPA (Ingenuity Pathway Analysis) of proteins enriched in Halo-SETD2C purification. AP-MS affinity purification-mass spectrometry. f Microscopy images showing localization of mCherry-hnRNP L and GFP-SETD2C fragment. The scale bar is 10 µm. GFP green fluorescent protein. The experiment was repeated at least four times all yielding similar results. g Halo purification was performed from extracts of 293T cells expressing Halo-SETD2C. Input and eluted samples were resolved on a gel and probed with an anti-hnRNP L antibody. The experiment was repeated at least two times all yielding similar results.

Article Snippet: hnRNP L and SETD2 human ORF were procured from Promega.

Techniques: Affinity Purification, Microscopy, Purification, Expressing, Silver Staining, Marker, Protein-Protein interactions, Mass Spectrometry

Journal: Nature Communications

Article Title: The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

doi: 10.1038/s41467-021-21663-w

Figure Lengend Snippet: RNase treatment was not performed for these experiments. a , c , e , g Cartoon illustrating the SETD2 and ySet2 constructs along with the known domains that were used in affinity-purifications. b , d , h Halo purification was performed from extracts of 293T cells expressing Halo- or Halo-FLAG-tagged proteins. Input and eluted samples were resolved on gel followed by silver staining or western blotting. The expected band for the target proteins are depicted by arrows. HE high exposure, LE low exposure, * non-specific. The experiments were repeated at least two times all yielding similar results. f Table showing the dNSAFs of the listed proteins post mass spectrometry analysis of purified complexes obtained by affinity purification of Halo-Set2 from 239T extracts. AWS associated with SET, SET Su(var)3–9, Enhancer-of-zeste and Trithorax, SRI Set2-Rpb1 Interaction, dNSAF distributed normalized spectral abundance factor, NLS nuclear localization signal.

Article Snippet: hnRNP L and SETD2 human ORF were procured from Promega.

Techniques: Construct, Purification, Expressing, Silver Staining, Western Blot, Mass Spectrometry, Affinity Purification

Journal: Nature Communications

Article Title: The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

doi: 10.1038/s41467-021-21663-w

Figure Lengend Snippet: a , d Cartoon illustrating the hnRNP L and SETD2 constructs along with the known domains that were used in affinity-purifications and in vitro binding. b Halo purification was performed from extracts of 293T cells co-expressing Halo-tagged SETD2C and mCherry-HA-hnRNP L. Input and eluted samples were resolved on gel followed by western blotting. The expected band for the target proteins are depicted by arrows. RNase treatment was not performed for these experiments. The experiment was repeated at least two times all yielding similar results. c Microscopy images showing the localization of mCherry-hnRNP L constructs. The scale bar is 10 µm. The experiment was repeated at least four times all yielding similar results. e GST pull-down was performed using recombinant proteins purified from bacteria. RNase was included in the binding assay. The input and eluted samples were resolved on gel followed by western blotting with the depicted antibodies. The experiment was repeated at least two times all yielding similar results. AWS associated with SET, SET Su(var)3–9, Enhancer-of-zeste and Trithorax, SRI Set2-Rpb1 interaction, SHI SETD2-hnRNP interaction, RRM RNA-recognition motif, NLS nuclear localization signal, GST glutathione-S-transferase.

Article Snippet: hnRNP L and SETD2 human ORF were procured from Promega.

Techniques: Construct, In Vitro, Binding Assay, Purification, Expressing, Western Blot, Microscopy, Recombinant, Bacteria

Journal: Nature Communications

Article Title: The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

doi: 10.1038/s41467-021-21663-w

Figure Lengend Snippet: a , b , e Heat maps showing the enrichment of pathways in the IPA (Ingenuity Pathway Analysis) and proteins in MudPIT analysis. c , d GO-term analysis of proteins using ShinyGO ( http://bioinformatics.sdstate.edu/go/ ) identified by MudPIT in the affinity purification of SETD2 SHI and 1964–2113 fragments. f Chart showing the enriched pathways in IPA of ySet2 proteins. SRI Set2-Rpb1 interaction, SHI SETD2-hnRNP interaction.

Article Snippet: hnRNP L and SETD2 human ORF were procured from Promega.

Techniques: Affinity Purification

Journal: Nature Communications

Article Title: The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

doi: 10.1038/s41467-021-21663-w

Figure Lengend Snippet: a RNA was isolated from 293T cells transfected with siRNA and RTPCR was performed to check transcript levels. gapdh was used as a normalization control. Western blot of whole-cell lysates was performed with the depicted antibodies. The experiment was repeated at least seven times all yielding similar results. b Chart showing the decrease in expression of the genes depicted based on RNA-seq analysis post siRNA treatment. c , e , f Pie charts showing the fractions of differentially expressed genes and AS events that occur in both setd2 and hnrnpl depletion. d Heat map showing the genes that show differential expression in both setd2 and hnrnpl depleted 293T cells. AS alternative splicing, A3SS alternate 3′ splice site, A5SS alternate 5′ splice site, RI retained intron, MXE mutually exclusive exon, SE skipped exon.

Article Snippet: hnRNP L and SETD2 human ORF were procured from Promega.

Techniques: Isolation, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, RNA Sequencing, Quantitative Proteomics, Alternative Splicing

Journal: Nature Communications

Article Title: The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

doi: 10.1038/s41467-021-21663-w

Figure Lengend Snippet: a Cartoon illustrating the SETD2 constructs along with their known domains that were used to compare the ability to deposit H3K36me3 in KO cells. b , c Western blot with the depicted antibodies of whole-cell lysates of KO cells expressing SETD2 mutants. The experiment was repeated at least five times all yielding similar results. d Bar graph showing H3 normalized H3K36me3 signal intensity of data depicted in ( c ). n = 4 independent biological samples examined in four independent experiments. Unpaired t test (two-tailed) was performed. p -value <0.05 (FLΔSRI vs FL = 0.0071; FLΔSHI vs FL = 0.0344; FLΔSRIΔSHI vs FL = 0.0013) was considered significant. Data are presented as mean values with standard error of mean. e , f , g , h Metagene plot and boxplot depicting the distribution of H3K36me3 upon expression of SETD2 FL, FLΔSRI, and FLΔSHI in setd2Δ 293T cells. For each sample n = 2 independent biological samples examined in the same sequencing run. In the boxplots, the black line inside the box shows the median. The box bottom and top border correspond to 25th and 75th percentiles (Q1 and Q3, respectively). The whiskers represent ranges from Q1 − 1.5 * IQR to Q3 + 1.5 * IQR where IQR stands for interquartile range (Q3–Q1). Data points outside the whiskers could be outliers and are marked as black dots. AWS associated with SET, SET Su(var)3–9, Enhancer-of-zeste and Trithorax, SRI Set2-Rpb1 interaction, SHI SETD2-hnRNP interaction, NLS nuclear localization signal, KO knock out ( setd2Δ 293T cells), TSS transcription start site, TES transcription end site.

Article Snippet: hnRNP L and SETD2 human ORF were procured from Promega.

Techniques: Construct, Western Blot, Expressing, Two Tailed Test, Sequencing, Knock-Out