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: Cell

Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

doi: 10.1016/j.cell.2019.04.035

Figure Lengend Snippet:

Article Snippet: Model-based Analysis of Genomewide CRISPR/Cas9 Knockout (MAGeCK) , , https://sourceforge.net/p/mageck/wiki/Home.

Techniques: Virus, Isolation, Recombinant, Transfection, Membrane, Sequencing, Software, CRISPR, Knock-Out

Journal: eLife

Article Title: Rare missense variants in the human cytosolic antibody receptor preserve antiviral function

doi: 10.7554/eLife.48339

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Alt-R CRISPR-Cas9 tracrRNA , IDT , Cat#: 1072532 , .

Techniques: Knock-Out, CRISPR, Gene Knockout, Recombinant, Construct, Plasmid Preparation, Western Blot, Sequencing, Clone Assay, Purification, Gel Extraction, Transfection, Software, Reporter Gene Assay

Journal: Oncogenesis

Article Title: Androgen receptor variant 7 exacerbates hepatocarcinogenesis in a c-MYC-driven mouse HCC model

doi: 10.1038/s41389-023-00449-3

Figure Lengend Snippet: A RNA-seq transcriptome analysis showed that 150 genes that were upregulated by c-MYC were repressed by AR-FL and/or AR-V7 co-expression; and 31 genes that were downregulated by c-MYC were upregulated by AR-FL and/or AR-V7 co-expression in both male (left) and female (right) mice at 20 dpi respectively. Each blue line represents differential expression levels (fold change in reference to control, Y axis) in the tumors of the respective combination of injected gene(s), X axis. Red lines highlight the differential expression levels of Cldn7 in respective samples. B Quantitative RT-PCR for expressions of Cldn7 in respective groups at 20 dpi. Expression of Cldn7 in male mice of c-MYC/p53KO (hep-c-MYC/p53KO) tumor at 11 dpi (pink bar), indicating that Cldn7 was repressed in p53-deficient conditions and hence was likely regulated by p53 [70–72]. C Expression of p53 in respective mouse groups in male and female, indicating p53 was upregulated by c-MYC but not significantly affected by AR-FL or AR-V7 co-expression. D Immunofluorescent images of c-MYC (red), Cldn7 FLAG (green), and DNA (blue) in the liver of hep-c-MYC/p53KO/Cldn7 FLAG mouse at 2 dpi, showing co-expression of the respective transgenes in the same cells. Scale bar = 50 µm E , Macroscopic phenotypes of the livers in male mice at 11 dpi injected with the expression vectors of c-MYC (c-MYC) ( n = 5), c-MYC under p53-deficient condition (c-MYC/p53KO) ( n = 6), or c-MYC and Cldn7 FLAG under p53-deficient condition (c-MYC/p53KO/Cldn7 FLAG ) ( n = 5). F Liver to body weight ratio of the samples presented in E , indicating that the exacerbation of c-MYC-driven HCC under p53-deficient conditions was completely reversed by co-expression of Cldn7 FLAG . Statistical significance by one-way ANOVA with Tukey’s multiple comparisons test for B , C , and F ; * p < 0.05; ** p < 0.001; *** p < 0.0001. Error bars indicate mean ± SEM.

Article Snippet: The pT3-based c-MYC expression vector pT3-c-MYC (#92046) [ ], the SB100 transposase expression vector pCMV-SB100 (#34879) [ ], the CRISPR/Cas9-base mouse p53 knockout vector pX330-p53 (#59910) [ ], and the full-length human androgen receptor vector pLENTI6.3/AR-GC-E2325 (#85128) were obtained from the Addgene Repository (Watertown, MA).

Techniques: RNA Sequencing, Expressing, Quantitative Proteomics, Control, Injection, Quantitative RT-PCR

Journal: Oncogenesis

Article Title: Androgen receptor variant 7 exacerbates hepatocarcinogenesis in a c-MYC-driven mouse HCC model

doi: 10.1038/s41389-023-00449-3

Figure Lengend Snippet: AR-V7 potentiates the cancer-related genes and oncogenic processes regulated by c-MYC, while it represses a tumor suppressor Cldn7 that is upregulated by c-MYC-overexpression via its activation of the p53 signaling pathway. AR-FL partly exerts similar functions to those of AR-V7 in the presence of androgen in males.

Article Snippet: The pT3-based c-MYC expression vector pT3-c-MYC (#92046) [ ], the SB100 transposase expression vector pCMV-SB100 (#34879) [ ], the CRISPR/Cas9-base mouse p53 knockout vector pX330-p53 (#59910) [ ], and the full-length human androgen receptor vector pLENTI6.3/AR-GC-E2325 (#85128) were obtained from the Addgene Repository (Watertown, MA).

Techniques: Over Expression, Activation Assay

Journal: Parasites & Vectors

Article Title: csi-miR-96-5p delivered by Clonorchis sinensis extracellular vesicles promotes intrahepatic cholangiocarcinoma proliferation and migration via the ferroptosis-related PTEN/SLC7A11/GPX4 axis

doi: 10.1186/s13071-023-06075-7

Figure Lengend Snippet: PTEN inhibited ICC proliferation and migration via a ferroptosis mechanism. a Stable PTEN overexpression (PTEN-EXO) or CRISPR/Cas9-based knockout (PTEN-KO) HuCCT1 cell lines were established; the scale shown is 100 um. b Cell proliferation was measured by CCK-8 assay in vitro. c Cell proliferation was measured by tumor xenograft in vivo. d Cell migration was measured by transwell assay. e Cell GSH/GSSG radio assay. f Cell Fe 2+ assay. g Cell MDA assay. h , i Cell WB assay of SLC7A11 and GPX4. ICC , intrahepatic cholangiocarcinoma; WB , western blotting; GSH , glutathione; GSSG , GSH disulfide; MDA , malondialdehyde. * P < 0.05, ** P < 0.01

Article Snippet: To create stable cell lines, recombinant lentivirus (LV) vectors containing csi-miR-96-5p mimics (Shanghai Genchem Co., Ltd.), a PTEN overexpression plasmid (PTEN-EXO) (Sino Biological, China), and a PTEN CRISPR/Cas9 based knockout (PTEN-KO) plasmid (Shanghai Genchem Co., Ltd.) were constructed.

Techniques: Migration, Over Expression, CRISPR, Knock-Out, CCK-8 Assay, In Vitro, In Vivo, Transwell Assay, Multiple Displacement Amplification, Western Blot