Journal: Acta biochimica et biophysica Sinica

Article Title: Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

doi: 10.3724/abbs.2023037

Figure Lengend Snippet: Figure 1. Hsa_circ_0013880 and USP32 were highly expressed in bone marrow mononuclear cells (BMNCs) of AML patients (A) hsa_circ_ 0013880 is located at chr1: 145439592-145442628, and its gene symbol is thioredoxin interacting protein (TXNIP). (B) Convergent and divergent primers were used to verify the closed loop structure of hsa_circ_0013880. (C) Amplification of hsa_circ_0013880 and TXNIP after RNase R treatment. (D,E) The relative RNA/mRNA levels of hsa_circ_0013880 and USP32 were detected by real-time PCR. (F) Correlation of hsa_circ_0013880 and USP32 in AML patients. **P<0.01. Data are presented as the mean±SD. M, marker; HS, healthy samples.

Article Snippet: The antibodies used in this study are as follows: USP32 (1:500; Proteintech, Rosemont, USA), Cyclin D1 (1:400; ABclonal Technology, Wuhan, China), cleaved caspase-3 (1:500; Affbiotech, Changzhou, China), CDK4 (1:500; ABclonal Technology), P21 (1:1000; ABclonal Technology), cleaved caspase-9 (1:1000; CST, Danvers, USA), cleaved PARP (1:500; CST), Rap1b (1:1000; Proteintech), β-actin (Santa Cruz Biotechnology, Santa Cruz, USA), HRP-labelled goat anti-rabbit IgG (Beyotime Biotechnology), HRP-labelled goat anti-mouse IgG (Beyotime Biotechnology). β-Actin was used as the protein loading control.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Marker

Journal: Acta biochimica et biophysica Sinica

Article Title: Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

doi: 10.3724/abbs.2023037

Figure Lengend Snippet: Figure 2. Bioinformatics predictions and dual-luciferase validation assays performed on HL-60 cells (A) A dual-luciferase assay was performed to verify the binding between USP32 and miR-148a-3p or miR-20a-5p. (B,C) Real-time PCR was used to evaluate the knockdown efficiency of miR- 148a-3p or miR-20a-5p. (D,E) The mRNA level of USP32 was detected by real-time PCR following microRNA downregulation. (F) Dual-luciferase assay was performed to verify the binding between hsa-circ-0013880 and miR-148a-3p or miR-20a-5p. For dual-luciferase assay, 293T cells were cotransfected with pmirGLO-wt-hsa_circ_0013880, pmirGLO-mut-hsa_circ_0013880, pmirGLO-wt-USP32 and pmirGLO-mut-USP32, along with miR-148a-3p/NC mimics or miR-20a-5p/NC mimics. **P<0.01. Data are presented as the mean±SD. Ctrl, control parental cells.

Article Snippet: The antibodies used in this study are as follows: USP32 (1:500; Proteintech, Rosemont, USA), Cyclin D1 (1:400; ABclonal Technology, Wuhan, China), cleaved caspase-3 (1:500; Affbiotech, Changzhou, China), CDK4 (1:500; ABclonal Technology), P21 (1:1000; ABclonal Technology), cleaved caspase-9 (1:1000; CST, Danvers, USA), cleaved PARP (1:500; CST), Rap1b (1:1000; Proteintech), β-actin (Santa Cruz Biotechnology, Santa Cruz, USA), HRP-labelled goat anti-rabbit IgG (Beyotime Biotechnology), HRP-labelled goat anti-mouse IgG (Beyotime Biotechnology). β-Actin was used as the protein loading control.

Techniques: Luciferase, Biomarker Discovery, Binding Assay, Real-time Polymerase Chain Reaction, Knockdown, Control

Journal: Acta biochimica et biophysica Sinica

Article Title: Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

doi: 10.3724/abbs.2023037

Figure Lengend Snippet: Figure 3. Overexpression of hsa_circ_0013880 enhanced the malignant behaviors of HL-60 cells by increasing the expression of USP32 Cells were infected with hsa_circ_0013880-overexpressing lentiviruses (LV-Circ) and control lentiviruses (LV-vec) for 48 h. (A) Real-time PCR was performed to detect the expression of hsa_circ_0013880. (B,C) The relative expression of USP32 was detected by real-time PCR and western blot analysis. Next, cells were infected with LV-Circ or control LV-vec and USP32 shRNA lentiviruses (LV-USP32sh) or control shRNA lentiviruses (LV- shCtrl) for 48 h. (D) Cell viability was evaluated by CCK-8 assay. (E) Annexin V-APC/PI staining was used to quantitatively analyse early and late cell apoptosis (Q2 and Q4). (F) Western blot analysis of the expressions of Cyclin D1 and cleaved caspase-3 proteins. (G) Transwell migration assay. (H) Colony formation assay. *P<0.05 and **P<0.01. Data are presented as the mean±SD. Ctrl, control parental cells.

Article Snippet: The antibodies used in this study are as follows: USP32 (1:500; Proteintech, Rosemont, USA), Cyclin D1 (1:400; ABclonal Technology, Wuhan, China), cleaved caspase-3 (1:500; Affbiotech, Changzhou, China), CDK4 (1:500; ABclonal Technology), P21 (1:1000; ABclonal Technology), cleaved caspase-9 (1:1000; CST, Danvers, USA), cleaved PARP (1:500; CST), Rap1b (1:1000; Proteintech), β-actin (Santa Cruz Biotechnology, Santa Cruz, USA), HRP-labelled goat anti-rabbit IgG (Beyotime Biotechnology), HRP-labelled goat anti-mouse IgG (Beyotime Biotechnology). β-Actin was used as the protein loading control.

Techniques: Over Expression, Expressing, Infection, Control, Real-time Polymerase Chain Reaction, Western Blot, shRNA, CCK-8 Assay, Staining, Transwell Migration Assay, Colony Assay

Journal: Acta biochimica et biophysica Sinica

Article Title: Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

doi: 10.3724/abbs.2023037

Figure Lengend Snippet: Figure 4. Silencing of USP32 inhibited the proliferation of AML cells HL-60 and U937 cells were infected with USP32 shRNA lentiviruses (LV- USP32sh#1 and LV-USP32sh#2) or control shRNA lentiviruses (LV-shCtrl) for 48 hours. (A,E) The mRNA levels of USP32 were detected by real-time PCR. (B,F) The expression of USP32 protein was analysed by western blot analysis. (C,G) Cell viability was evaluated by CCK-8 assay. (D,H) Colony formation assay was carried out after infection. (I) The cell cycle was determined by flow cytometry. (J) Western blot analysis of the expressions of Cyclin D1, CDK4 and P21 proteins. **P<0.01. Data are presented as the mean±SD. Ctrl, control parental cells.

Article Snippet: The antibodies used in this study are as follows: USP32 (1:500; Proteintech, Rosemont, USA), Cyclin D1 (1:400; ABclonal Technology, Wuhan, China), cleaved caspase-3 (1:500; Affbiotech, Changzhou, China), CDK4 (1:500; ABclonal Technology), P21 (1:1000; ABclonal Technology), cleaved caspase-9 (1:1000; CST, Danvers, USA), cleaved PARP (1:500; CST), Rap1b (1:1000; Proteintech), β-actin (Santa Cruz Biotechnology, Santa Cruz, USA), HRP-labelled goat anti-rabbit IgG (Beyotime Biotechnology), HRP-labelled goat anti-mouse IgG (Beyotime Biotechnology). β-Actin was used as the protein loading control.

Techniques: Infection, shRNA, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, CCK-8 Assay, Colony Assay, Flow Cytometry

Journal: Acta biochimica et biophysica Sinica

Article Title: Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

doi: 10.3724/abbs.2023037

Figure Lengend Snippet: Figure 5. Silencing of USP32 promoted the apoptosis of AML cells HL-60 and U937 cells were infected with USP32 shRNA lentiviruses (LV- USP32sh#1 and LV-USP32sh#2) or control shRNA lentiviruses (LV-shCtrl) for 48 hours. (A,B) Annexin V-APC/PI staining was used to quantitatively analyse early and late cell apoptosis (Q2 and Q4). (C) The protein levels of cleaved caspase-3, cleaved caspase-9 and cleaved PARP were analysed by western blot analysis. (D,E) The activity of caspase-3 and caspase-9 was estimated by cleavage of the corresponding Ac-DEVD-pNA or Ac-LEVD- pNA to pnitroanilide (pNA). **P<0.01. Data are presented as the mean±SD. Ctrl, control parental cells.

Article Snippet: The antibodies used in this study are as follows: USP32 (1:500; Proteintech, Rosemont, USA), Cyclin D1 (1:400; ABclonal Technology, Wuhan, China), cleaved caspase-3 (1:500; Affbiotech, Changzhou, China), CDK4 (1:500; ABclonal Technology), P21 (1:1000; ABclonal Technology), cleaved caspase-9 (1:1000; CST, Danvers, USA), cleaved PARP (1:500; CST), Rap1b (1:1000; Proteintech), β-actin (Santa Cruz Biotechnology, Santa Cruz, USA), HRP-labelled goat anti-rabbit IgG (Beyotime Biotechnology), HRP-labelled goat anti-mouse IgG (Beyotime Biotechnology). β-Actin was used as the protein loading control.

Techniques: Infection, shRNA, Control, Staining, Western Blot, Activity Assay

Journal: Acta biochimica et biophysica Sinica

Article Title: Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

doi: 10.3724/abbs.2023037

Figure Lengend Snippet: Figure 6. Silencing USP32 could upregulate the ubiquitination level of Rap1b in AML cells (A) HL-60 and U937 cells were infected with USP32 shRNA lentiviruses (LV-USP32sh#1 and LV-USP32sh#2) or control shRNA lentiviruses (LV-shCtrl) for 48 h. The protein expression of Rap1b was analysed by western blot analysis. (B) Co-IP analyses of USP32 and Rap1b proteins in HL-60 and U937 cells. (C) Western blot analysis of Rap1b expression in AML cells infected with the indicated lentiviral particles and exposed to CHX or treated with MG132 and CHX for 8 h. (D) Ubiquitination assay of Rap1b in HL-60 and U937 cells infected with the indicated lentiviral particles. *P<0.05 and **P<0.01. n.s., no significance. Data are presented as the mean±SD. Ctrl, control parental cells.

Article Snippet: The antibodies used in this study are as follows: USP32 (1:500; Proteintech, Rosemont, USA), Cyclin D1 (1:400; ABclonal Technology, Wuhan, China), cleaved caspase-3 (1:500; Affbiotech, Changzhou, China), CDK4 (1:500; ABclonal Technology), P21 (1:1000; ABclonal Technology), cleaved caspase-9 (1:1000; CST, Danvers, USA), cleaved PARP (1:500; CST), Rap1b (1:1000; Proteintech), β-actin (Santa Cruz Biotechnology, Santa Cruz, USA), HRP-labelled goat anti-rabbit IgG (Beyotime Biotechnology), HRP-labelled goat anti-mouse IgG (Beyotime Biotechnology). β-Actin was used as the protein loading control.

Techniques: Ubiquitin Proteomics, Infection, shRNA, Control, Expressing, Western Blot, Co-Immunoprecipitation Assay

Journal: Acta biochimica et biophysica Sinica

Article Title: Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

doi: 10.3724/abbs.2023037

Figure Lengend Snippet: Figure 7. USP32 inhibits the growth of HL-60 cells via regulation of Rap1b (A,B) HL-60 cells were infected with Rap1b-overexpressing lentiviruses (LV-Rap1b) or control lentiviruses (OE-vec) for 48 h. The relative expression of Rap1b was detected by real-time PCR and western blot analysis. Furthermore, cells were infected with LV-Rap1b or OE-vec and USP32 shRNA lentiviruses (LV-USP32sh) or control shRNA lentiviruses (LV-shCtrl) for 48 hours. (C) CCK8 assay was performed to evaluate cell viability. (D) Annexin V-APC/PI staining was used to quantitatively analyse early and late cell apoptosis (Q2 and Q4). (E,F) The protein levels of Rap1b, Cyclin D1 and cleaved caspase-3 were analysed by western blot analysis. *P<0.05 and **P<0.01. Data are presented as the mean±SD. Ctrl, control parental cells.

Article Snippet: The antibodies used in this study are as follows: USP32 (1:500; Proteintech, Rosemont, USA), Cyclin D1 (1:400; ABclonal Technology, Wuhan, China), cleaved caspase-3 (1:500; Affbiotech, Changzhou, China), CDK4 (1:500; ABclonal Technology), P21 (1:1000; ABclonal Technology), cleaved caspase-9 (1:1000; CST, Danvers, USA), cleaved PARP (1:500; CST), Rap1b (1:1000; Proteintech), β-actin (Santa Cruz Biotechnology, Santa Cruz, USA), HRP-labelled goat anti-rabbit IgG (Beyotime Biotechnology), HRP-labelled goat anti-mouse IgG (Beyotime Biotechnology). β-Actin was used as the protein loading control.

Techniques: Infection, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, shRNA, CCK-8 Assay, Staining

Journal:

Article Title: The Tre2 ( USP6 ) oncogene is a hominoid-specific gene

doi: 10.1073/pnas.0437015100

Figure Lengend Snippet: Schematic representation of the chimeric Tre2 coding sequence, showing domains derived from the USP32 and TBC1D3 parental genes. These regions of overlap are conserved in the genomic structure. The full-length Tre2, USP32, and TBC1D3 genes encode proteins of 1,406, 1,604, and 549 aa, respectively.

Article Snippet: A full-length clone of USP32 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF533230","term_id":"22532404","term_text":"AF533230"}} AF533230 ) was isolated from a brain cDNA library (OriGene Technologies, Rockville, MD).

Techniques: Sequencing, Derivative Assay

Journal:

Article Title: The Tre2 ( USP6 ) oncogene is a hominoid-specific gene

doi: 10.1073/pnas.0437015100

Figure Lengend Snippet: Human tissue expression pattern of Tre2, USP32, and TBC1D3. cDNA panels (CLONTECH) were screened by semiquantitative RT-PCR and are as follows: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, spleen; 10, thymus; 11, prostate; 12, testis; 13, ovary; 14, small intestine; 15, peripheral blood leukocyte; and 16, negative control. Specific RT-PCR products for Tre2, USP32, and TBC1D3 are designed by amplification across the unique juncture for the respective transcripts. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplification was used as a control.

Article Snippet: A full-length clone of USP32 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF533230","term_id":"22532404","term_text":"AF533230"}} AF533230 ) was isolated from a brain cDNA library (OriGene Technologies, Rockville, MD).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Amplification, Control

Journal:

Article Title: The Tre2 ( USP6 ) oncogene is a hominoid-specific gene

doi: 10.1073/pnas.0437015100

Figure Lengend Snippet: Hybridization of Tre2 domains to DNA from diverse species. (A) Probe derived from human Tre2 (Tre2-A) (nucleotides 1697–3193) containing the TBC domain homologous to TBC1D3. Rabbit DNA in lane 8 was obtained from a different cell line than the DNA in lane 5. (B) Probe derived from human Tre2 (Tre2-B) (nucleotides 3163–5947) containing the ubiquitin protease domain homologous to USP32. Hybridization of Tre2-A probe (C) and Tre2-B probe (D) using a panel of primate DNA samples. Lanes and designations are as follows: 1, human; 2, chimpanzee (Pan troglodyte); 3, bonobo (Pan paniscus); 4, lowland gorilla (Gorilla gorilla); 5, orangutan (Pongo pygmaeus; overloaded); 6, rhesus monkey (Macaca mulatta); 7, pigtailed macaque (Macaca nemestrina); 8, red-bellied tamarin (Saguinus labiatus); 9, black-handed spider monkey (Ateles geoffroyi); 10, wooly monkey (Lagothrix lagotricha); 11, ring-tailed lemur (Lemur catta); and 12, mouse (Mus musculus; negative control).

Article Snippet: A full-length clone of USP32 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF533230","term_id":"22532404","term_text":"AF533230"}} AF533230 ) was isolated from a brain cDNA library (OriGene Technologies, Rockville, MD).

Techniques: Hybridization, Derivative Assay, Ubiquitin Proteomics, Negative Control

Journal:

Article Title: The Tre2 ( USP6 ) oncogene is a hominoid-specific gene

doi: 10.1073/pnas.0437015100

Figure Lengend Snippet: (A) Schematic representation of PCR fragments amplified for specific analysis of the Tre2 and USP32 sequences. Homologous exons were amplified in the PCRs (Tre2, exon 15 and USP32, exon 19). (B) PCR amplification of genomic DNA used to test for the presence of junctures specific to Tre2 and USP32. Lanes and designations are as follows: 1, human; 2, chimpanzee; 3, bonobo; 4, lowland gorilla; 5, orangutan; 6, rhesus monkey; 7, pigtailed macaque; 8, red-bellied tamarin; 9, black-handed spider monkey; 10, wooly monkey; 11, no template control. The 3.2-kb band seen in lane 8 occurs because of an insertion of a nonfunctional pseudogene within the intronic sequence of USP32. (C) Phylogenetic (maximum likelihood) tree of Tre2 and USP32 shared DNA sequences. Analysis was performed on ≈940 bp. Analysis included intron and exon sequences. The tree was rooted with New World monkeys as an outgroup. Arrows indicate the formation of the novel gene Tre2 in hominoids and the expansion of the segmental duplication. hum, human; bono, bonobo; chimp, chimpanzee; gor, lowland gorilla; orang, orangutan; mac, pigtailed macaque; rhes, rhesus monkey; tam, red-bellied tamarin; spid, black-handed spider monkey; wool, wooly monkey.

Article Snippet: A full-length clone of USP32 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF533230","term_id":"22532404","term_text":"AF533230"}} AF533230 ) was isolated from a brain cDNA library (OriGene Technologies, Rockville, MD).

Techniques: Amplification, Control, Sequencing