setd2 construct  (OriGene)

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: SETD2 deficiency in advanced phases of CML. (A) Representative Western blot results for SETD2 protein and H3K36me3 levels in CML patients as compared with a pool of healthy donors (HDs). One of three independent experiments is shown. (B) Scatter plot of SETD2 levels estimated by densitometric analysis of Western blots. The median is indicated for each group. SETD2 signal intensities in single blots obtained from three individual experiments were normalized to those of beta‐actin and averaged. Normalized SETD2 levels calculated in CML patients were then expressed in comparison to normalized SETD2 levels detected in a pool of HDs, conventionally set to 1. The Student's t ‐test was used to compare the means between two groups (CP vs. HDs; mut‐CP vs. HDs; AP vs. HDs; my‐BC vs. HDs and finally ly‐BC vs. HDs). *** p < .0009; **** p < .0001.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

Techniques: Western Blot, Comparison

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: Effects of proteasomal inhibition on SETD2 expression and function. Co‐immunoprecipitation assays show that (A) in samples from SETD2‐deficient patients, bortezomib treatment rescued a hyper‐ubiquitinated SETD2 protein; (B) in K562 cells showing low basal SETD2 levels, bortezomib treatment increased SETD2 protein levels. SETD2 appears to be ubiquitinated and to bind MDM2 in the same context. A negative control (NC) obtained by immunoprecipitation of protein lysates using a resin conjugated with an anti‐IgG1 antibody was loaded for each immunoprecipitation experiment. One of three independent experiments is shown in all panels.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

Techniques: Inhibition, Expressing, Immunoprecipitation, Negative Control

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: Effects of MDM2 inhibition on SETD2 expression and function. Western blotting and co‐immunoprecipitation assays show that (A) silencing or (B) inhibiting MDM2 by siRNA (for 72 h) or by SP141 (5 µM for 24 h), respectively, rescued SETD2/H3K36me3 expression in K562 cells. A negative control (NC) obtained by the immunoprecipitation of protein lysates using a resin conjugated with an anti‐IgG1 antibody was loaded for each immunoprecipitation experiment. One of three independent experiments is shown in all panels.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

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

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: Aurora‐A kinase interacts with SETD2 and its inhibition affects SETD2 expression and function. Western blotting and co‐immunoprecipitation assays show that (A) proteasomal inhibition by carfilzomib (5 nM for 24 h) rescuing SETD2 expression reveals its interaction with Aurora‐A kinase; (B) both siRNA‐mediated silencing of Aurora‐A for 72 h and Aurora‐A inhibition by alisertib (100 nM for 24 h) rescued SETD2/H3K36me3 expression in K562 cells and resulted in SETD2 release from Aurora‐A kinase and SETD2 dephosphorylation on Ser/Thr residues. A negative control (NC) obtained by the immunoprecipitation of protein lysates using a resin conjugated with an anti‐IgG1 antibody was loaded for each immunoprecipitation experiment. One of three independent experiments is shown in all panels.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

Techniques: Inhibition, Expressing, Western Blot, Immunoprecipitation, De-Phosphorylation Assay, Negative Control

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: Effects of SETD2 expression on HR repair activation. (A–D) Immunofluorescence analysis of phosphorylated histone 2A.X (P‐H2AX(S139), green) and Rad51 (red) in SETD2‐deficient and SETD2‐proficient cells after sub‐lethal DNA damage induction by hydrogen peroxide exposure. Staining with DAPI (4′,6‐diamidino‐2‐phenylindole) indicates the nuclear localization of P‐H2AX(S139) and Rad51. (A) KCL22 (SETD2‐deficient) cells; (B) KCL22 tsSETD2 cells; (C) LAMA84 (SETD2‐proficient) cells; (D) LAMA84 silenced for SETD2. Scale bar: 100 µM. (E) Western blotting showed that SETD2 loss impairs ATM signalling pathway activation; in contrast, SETD2 forced expression restores the ability to induce ATM phosphorylation and subsequent p95 and H2AX activation, associated with an increase in CtIP expression.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

Techniques: Expressing, Activation Assay, Immunofluorescence, Staining, Western Blot, Phospho-proteomics

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: Effects of SETD2 loss of function on MMEJ activation. (A–D) Immunofluorescence analysis of cleaved‐PARP (green) and XRCC1 (red) in SETD2‐deficient and SETD2‐proficient cells after hydrogen peroxide exposure. Staining with DAPI (4′,6‐diamidino‐2‐phenylindole) indicates the nuclear localization of XRCC1 and cleaved‐PARP. (A) KCL22 (SETD2‐deficient) cells; (B) KCL22 tsSETD2 cells; (C) LAMA 84 cells silenced for SETD2; (D) LAMA 84 (SETD2‐proficient) cells. Scale bar: 100 µM.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

Techniques: Activation Assay, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: Effects of SETD2 loss of function on MMR activation. (A, B) Immunofluorescence analysis of MSH6 (red) and THEX1 (green) in SETD2‐deficient and SETD2‐proficient cells after hydrogen peroxide exposure. Staining with DAPI (4′,6‐diamidino‐2‐phenylindole) indicates the nuclear localization of MSH6 and THEX1. (A) LAMA84 parental cells versus LAMA84 cells silenced for SETD2; (B) KCL22 cells versus KCL22 SETD2‐transfected cells. Scale bar: 100 µM.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

Techniques: Activation Assay, Immunofluorescence, Staining, Transfection

Journal: Clinical and Translational Medicine

Article Title: SETD2 loss of function is a recurrent event in advanced‐phase chronic myeloid leukemia and contributes to genomic instability

doi: 10.1002/ctm2.70163

Figure Lengend Snippet: Comparison between SETD2/H3K36Me3 levels at diagnosis and progression and druggability of SETD2/H3K36me3 deficiency in cell lines and primary samples. (A) Western blot results for SETD2 protein and H3K36me3 levels in paired samples collected at diagnosis (D) and after progression (P) from 5 CML patients. Protein expression in patient 2 is not detectable (ND). One of three independent experiments is shown. (B) Flow cytometry analysis of Annexin‐V/PI positive cells. LAMA84 before and after SETD2 silencing, K562‐S and K562‐R cells were treated with 10 nM bortezomib, 40 nM ixazomib, 5 nM carfilzomib, 500 nM of alisertib and 5 µM SP141 for 24 h. Statistical analysis was performed by using dedicated software (GraphPad Prism 8.0). p ‐values were always <.0021. (C) Dose‐dependent inhibition of LAMA84 before and after SETD2 silencing, K562‐S and K562‐R cells in 10‐day methylcellulose colony‐forming assays. Cells were treated with increasing concentrations of bortezomib, ixazomib, and carfilzomib (proteasome inhibitors). Bortezomib treatment is represented by the blue line, carfilzomib is represented by the red line, and ixazomib by the green line. (D) Dose‐dependent inhibition of the mononuclear fraction obtained by four CML BC pts showing high and low SETD2 levels in 14‐day methylcellulose colony‐forming assays. Cells were treated with increasing concentrations of bortezomib, ixazomib, carfilzomib (proteasome inhibitors), and SP141 (MDM2 inhibitor). Bortezomib treatment is represented by the blue line, carfilzomib is represented by the red line, ixazomib by the green line, and SP141 by the yellow line. (E) Flow cytometry analysis of Annexin‐V/PI positive cells. K562 (showing low SETD2 expression) and KCL22 (SETD2‐deficient) cells were treated with 500 nM of alisertib and 10 nM bortezomib for 24 h. The Student's t ‐test was used to compare the means obtained from three independent experiments between two groups (treated cells vs. untreated). p ‐values always <.0016. Western blot results performed in the same conditions showed marked differences in caspase 9 and 3 cleavage and Bax and phosphorylated H2AX expression levels after alisertib and bortezomib treatments. Beta‐actin was used as a loading control. One of three independent experiments is shown in all panels.

Article Snippet: Subsequently, 80 μL of the cell suspension containing 1 × 10 6 cells was mixed with 2 μg of a GFP‐tagged SETD2 construct (Origene) and electroporated in a .2 cm cuvette using the Nucleofector 2b device (Lonza) with the Lonza Cell Line Nucleofection Kit V according to the Lonza Amaxa 4D‐Nucleofector Protocol.

Techniques: Comparison, Biomarker Discovery, Western Blot, Expressing, Flow Cytometry, Software, Inhibition, Control

Journal: bioRxiv

Article Title: The CNS Microenvironment Promotes Leukemia Cell Survival by Disrupting Tumor Suppression and Cell Cycle Regulation in Pediatric T-cell Acute Lymphoblastic Leukemia

doi: 10.1101/2023.09.01.555887

Figure Lengend Snippet: A) RNA-sequencing analysis of normalized FPKM values showing decreased expression of SETD2 in CNS3 diagnosis patients compared to CNS1 diagnosis samples. (Significance calculated by two-tailed, unpaired Student’s t-test. *p<0.05). B) RNA-sequencing correlation analysis of SETD2 and TP53 (left graph) showing a negative correlation (r=-0.694, **p=<0.01) and MDM2 (right graph) showing a positive correlation (r=0.628, *p=<0.05) in CNS relapsed T-ALL patients. (Correlation was calculated using Pearson correlation coefficients, two-tailed with 95% confidence interval). C) String diagram showing protein-protein interactions directly and indirectly experimentally linking SETD2 with TP53, MDM2, BCLX, BCL2, NANOG and OCT4. D) mRNA expression levels (fold change versus empty vector control) of MDM2, TP53, MYCC, OCT4, NANOG, BCL2 and BCLX 24 hours after SETD2 overexpression by transfection in MOLT16 cells. E) mRNA expression levels of SETD2 overexpression in T.-ALL cell lines MOLT16, Jurkat, CCL-119 negatively correlates with BCL2 (r=0.-995, p=0.066), BCLX (r=-0.969), MDM2 (r=-979) and TP53 (r=-0.993, p=0.075). (Correlation was calculated using Pearson correlation coefficients, two-tailed with 95% confidence interval).

Article Snippet: DH5α competent cells (Invitrogen) were transformed by heat shock in 42°C water bath using SETD2 Human Tagged ORF Clone (Origene, RG224760) or pCMV6□AC□GFP Mammalian Expression Vector (Origene, PS100010) followed by overnight culture on LB agar plates (Sigma-Aldrich) containing 100μg/ml ampicillin (Sigma-Aldrich) at 37°C.

Techniques: RNA Sequencing, Expressing, Biomarker Discovery, Two Tailed Test, Protein-Protein interactions, Plasmid Preparation, Control, Over Expression, Transfection