Journal: Cancer Cell International

Article Title: Silencing Uracil-DNA glycosylase inhibits colorectal cancer progression

doi: 10.1186/s12935-025-04089-y

Figure Lengend Snippet: UNG activated several signaling pathways in CRC cells. Western blot was performed to detect the levels of mTOR, p-mTOR, p70 S6K, p-P70 S6K, AKT, p-AKT, AMPK, p-AMPK, ERK, p-ERK, Bax, Bcl2, cleavages of caspase-9, and caspase-3 in UNG-knockdown CRC cells. Fold changes (Fc) are shown below the bars

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: UNG, P70 S6K, p-P70 S6K(S424) (1:1000, Bioworld), β-actin (1:1000, Abcam), cleaved caspase-3, AMPKα1/AMPKα2, p-AMPKα1(Thr183)/AMPKα2(Thr172) (1:1000, Beyotime), BAX, BCL2, AKT1/2/3, mTOR, and p-mTOR (Ser2448) (1:1000; BOSTER), cleaved caspase-9, p-AKT(Ser473), ERK1/2, and p-ERK1/2(Thr202/Tyr204)/(Thr185/Tyr187) (1:1000; ZEN BIO), and GAPDH (1:1000; Proteintech).

Techniques: Protein-Protein interactions, Western Blot, Knockdown

Journal: Asian journal of andrology

Article Title: CHD1 deletion stabilizes HIF1α to promote angiogenesis and glycolysis in prostate cancer.

doi: 10.4103/aja202287

Figure Lengend Snippet: Figure 3: CHD1 deletion stabilizes HIF1α via PHD2. (a) Expression of key proteins involved in HIF1α degradation in ARCaPE and DU145 cells after CHD1 knockout. (b) PHD2 mRNA levels in ARCaPE and DU145 cells after CHD1 knockout. *P < 0.05. (c) Hydroxylation of HIF1α in ARCaPE and DU145 cells after CHD1 knockout. (d) Luciferase reporter assay of ODD domain degradation in ARCaPE and DU145 cells after CHD1 knockout. *P < 0.05. (e) Diagram of the CRISPR-resistant CHD1 overexpression system. The CRISPR-targeted CHD1 sequence was mutated to escape recognition by the gRNA, but the amino acid sequence was not changed. The mutated CHD1 cDNA was cloned into a lentiviral expression vector. (f) Expression of PHD2 and HIF1α after overexpression of CRISPR-resistant CHD1 in ARCaPE and DU145 cells with CHD1 knockout. HIF1α: hypoxia-inducible factor 1α; CHD1: chromodomain-helicase-DNA-binding protein 1; PHD2: prolyl hydroxylase domain protein 2; ODD: oxygen-dependent degradation; CRISPR: clustered regularly interspaced palindromic repeat; Cas9: CRISPR-associated protein 9; gRNA: guide RNA; cDNA: complementary DNA; KO: knockout; p-eIF2α: phosphorylated eukaryotic translation initiation factor 2 subunit alpha; VHL: von Hippel–Lindau tumor suppressor; IB: immunoblotting; IP: immunoprecipitation.

Article Snippet: Clustered regularly interspaced palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9)-based CHD1 knockout CRISPR/Cas9-based CHD1 knockout was conducted with a Cas9 lentiviral vector (#71489, Addgene, Watertown, MA, USA) and a guide RNA (gRNA) vector (#84752, Addgene).

Techniques: Expressing, Knock-Out, Luciferase, Reporter Assay, CRISPR, Over Expression, Sequencing, Clone Assay, Plasmid Preparation, Binding Assay, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Schematic representation of generation of Tet2 -knockout clones with CRISPR/Cas9. ( B ) Proliferative outgrowth of Cebpa p30/p30 cells with Tet2 indels. ( C ) Volcano plot depicting differentially expressed genes dependent on the Tet2 mutational status in Cebpa p30/p30 cells (n=5–7 per group). ( D ) Experimental setup for evaluating the effect of Tet2 -deficiency ( Tet2 −/− ) in Cebpa DM AML initiation in vivo . ( E ) Myeloid (Mac1 + ) contribution of donor-derived blood and bone marrow (BM) cells evaluated at 12, 24, and 36 weeks after BM transplantation and Cre-LoxP recombination to generate a Cebpa −/p30 and Tet2 −/− hematopoietic compartment (n=3–6 per genotype and timepoint). ( F ) Survival of lethally irradiated recipient mice after BM transplantation and Cre-LoxP recombination (n=12–14/group). ( G ) Volcano plot depicting differentially expressed genes dependent on Tet2 deficiency status in Cebpa −/p30 leukemic blasts (n=3 per group). ( H ) Frequency of proliferating (Ki67 + ) cells in BM of moribund recipient mice (n=3 per group). **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knock-Out, Clone Assay, CRISPR, In Vivo, Derivative Assay, Transplantation Assay, Irradiation

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Experimental setup for evaluating the effect of Gata2 knockdown, via short hairpin RNA (shRNA) mediated silencing, on Cebpa p30/p30 leukemic cells in a competitive in vivo assay. ( B ) Gata2 mRNA in Cebpa p30/p30 leukemic cells prior to transplantation. ( C ) Representative flow cytometry profiles of input and output of shControl (no knockdown), sh Gata2 A (low knockdown), and sh Gata2 D (high knockdown). ( D ) Competitive advantage of targeting shRNA (GFP + ) vs. non-targeting shRNA (YFP + ) cells in vivo assessed as by flow cytometry (n=3–4 per group). ( E ) Experimental setup for Gata2 CRISPR/Cas9 mutagenesis in Cebpa p30/p30 cells, and outgrowth of heterozygous mutated clones. Percentages of Gata2 mutated clones are indicated. ( F ) Growth curve of Cebpa p30/p30 clones with Gata2 mutation ( Cebpa p30/p30 Gata2 +/MUT ) or wild type Gata2 ( Cebpa p30/p30 Gata2 +/+ ). Red lines mark individual clones. (G) Presence or absence of GATA2 mutations ( GATA2 MUT ) in CEBPA double mutated ( CEBPA DM ) AML cases (n=460) with or without TET2 mutations ( TET2 MUT ) in aggregated data from published cohorts – , , , . *=P<0.05, **=P<0.01

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knockdown, shRNA, In Vivo, Transplantation Assay, Flow Cytometry, CRISPR, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Gata2 mRNA expression in mouse Cebpa p30/p30 leukemic granulocyte/monocyte progenitors (GMPs) vs normal GMPs and, ( B ) CEBPA binding to the Gata2 distal hematopoietic enhancer ( G2 DHE; −77kb) region, data from Jakobsen et al. (n=2–4 per group). ( C ) Schematic genomic view of the Gata2 distal hemeatopoietic enhancer ( G2 DHE), including tracks from CEBPA and H3K27Ac chromatin immunoprecipitation sequencing (ChIP-seq) in Cebpa p /p30 cells (data from Heyes et al. ), TET2 ChIP-seq in AML-ETO expressing cells (data from Rasmussen et al. ),Targeting of the G2 DHE by dual-( D ) or single-( E ) guided CRISPR-Cas9 in Cebpa p30/p30 cells using indicated sgRNAs (n=3/condition). ( F ) Experimental setup for evaluating the effects of Cebpa knockout on Gata2 V2 mRNA expression and DNA methylation of the CpG island at the promoter of Gata2 V2 in MLL-fusion driven AML ( iMLL-AF9 ). ( G ) Cebpa and ( H ) Gata2 V2 mRNA expression upon induction of Cre-LoxP recombination and, ( I ) DNA methylation of the Gata2 V2 promoter CpG-island (2 biological replicates per genotype). ( J ) Frequency of GATA2 and/or TET2 mutations ( GATA2 MUT and TET2 MUT , respectively) in CEBPA high expressing ( CEBPA HIGH n=45) vs. CEBPA low expressing (CEBPA LOW n=61) AML cases, data from Beat AML cohort . *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Expressing, Binding Assay, ChIP-sequencing, CRISPR, Knock-Out, DNA Methylation Assay