Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-specific protease 21 is critical for cancer cell mitochondrial function and regulates proliferation and migration

doi: 10.1016/j.jbc.2024.107793

Figure Lengend Snippet: Analysis of STAT3-dependent transcriptome and proteome changes in HAP-1 USP21 KO versus HAP-1 WT cells. Comparative transcriptomic and proteomic analysis of USP21 KO HAP-1 cells versus control cells. Volcano plots show differentially expressed genes ( A ) and proteins ( B ) in USP21 KO HAP-1 cells versus parental HAP-1 cells. The data points above the significance threshold (q < 0.05) are marked in red (upregulated in USP21 KO) and blue (downregulated in USP21 KO). The gray dashed vertical lines indicate the thresholds of |log2(FoldChange)| = 1 and |log2(FoldChange)| = 0.5 for transcriptomic and proteomic data, respectively. Transcripts and proteins with insignificant changes are depicted in gray . Transcription factors regulating expression of 20 most significantly downregulated transcripts and the most downregulated proteins were analyzed using ChIP Enrichment Analysis (ChEA) and Encode databases and the names are marked on volcano plots. Transcripts and proteins regulated by STAT3 are marked on volcano plots in green squares , regulated in STAT3-independent way are marked in gray squares . STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease

Article Snippet: pTF-STAT3-Luc reporter vector (BioCat) and pcDNA3.1-HA-USP21, or pcDNA3.1 plasmids were transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

Techniques: Control, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-specific protease 21 is critical for cancer cell mitochondrial function and regulates proliferation and migration

doi: 10.1016/j.jbc.2024.107793

Figure Lengend Snippet: USP21 is a positive regulator of STAT3 expression and phosphorylation in HAP-1 cells . The effect of USP21 KO ( A ) and overexpression ( B ) on STAT3 expression level and Tyr705 phosphorylation in HAP-1 cells was analyzed by Western blot and detection of pSTAT3, STAT3, USP21, and β-actin. A , HAP-1 cells (parental and USP21 KO, 1 × 10 6 /sample) were treated with 50 ng/ml IL-6 for up to 20 min. Then cell lysates were used for Western blot analysis. The histogram ( right panel ) depicts the levels of pSTAT3 in HAP-1 WT (red ) and HAP-1 USP21 KO ( blue ) determined through densitometry corrected for actin ( B ). For analysis of STAT3 phosphorylation in HAP-1 cells overexpressing USP21, cells were transfected with pcDNA3.1-USP21 vector (1 μg and 5 μg) by nucleofection. After 24 h cells were stimulated with IL-6 for 5 and 20 min. The level of pSTAT3, STAT-3, USP21, and β-actin was detected by Western blot. Histogram ( right panel ) represent relative mean band intensity of pSTAT3 for HAP-1 cells untransfected ( red ) and transfected with 1 μg ( blue ) and 5 μg ( gray ) of pcDNA3.1-USP21. The effect of USP21 overexpression on STAT3 activation analyzed by Dual-Glo luciferase system ( C ). pTF-STAT3-Luc, pcDNA3.1-HA-USP21, or pcDNA3.1 (EV; empty vector) plasmids were transfected in HEK293 cells for 8 h, followed by activation with IL-6 for 16 h. Then, the cells were subjected to luminescence measurement by using a Dual-Glo luminescence assay. Interaction of USP21 with STAT3 ( D ). HA-USP21 or STAT3 encoding plasmids were transfected into HEK293T for 48 h, and then cell lysates were prepared for coimmunoprecipitation and immunoblotting against specific antibodies as indicated. The standard deviation was determined from three independent repetitions. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns indicates no statistical difference ( p > 0.05). IL-6, interleukin 6; pSTAT3, Tyr705 phosphorylated signal transducer and activator of transcription 3; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.

Article Snippet: pTF-STAT3-Luc reporter vector (BioCat) and pcDNA3.1-HA-USP21, or pcDNA3.1 plasmids were transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

Techniques: Expressing, Over Expression, Western Blot, Transfection, Plasmid Preparation, Activation Assay, Luciferase, Luminescence Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-specific protease 21 is critical for cancer cell mitochondrial function and regulates proliferation and migration

doi: 10.1016/j.jbc.2024.107793

Figure Lengend Snippet: USP21 drives proliferation, migration, and influences STAT3 phosphorylation in A549 cells . Cell proliferation of A549 transfected with scramble siRNA-siCONTROL ( red line ) and siUSP21 ( blue line ), was analyzed by cell counting every day for 4 days. The number of cells was determined automatically using Luna cell counter ( A ). The ability of cells in culture to grow was tested in the colony formation assay ( B ). A549 cells treated with scramble siRNA and siUSP21 were placed 2 × 10 2 cells in 24-well plates, incubated for 10 days, and the cells were stained with 0.1% crystal violet and counted ( left panel ). The mean number of colonies from six wells for scramble siRNA ( red ) and siUSP21 ( blue ) treated cells are presented on the histogram ( right panel ). A wound healing assay was used to assess the migration ability of A549 cells (the scale bar represents 200 μm) ( C ). Western blot analysis of STAT3 phosphorylation in IL-6-stimulated USP21 silencing A549 cells ( D ). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. IL-6, interleukin 6; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.

Article Snippet: pTF-STAT3-Luc reporter vector (BioCat) and pcDNA3.1-HA-USP21, or pcDNA3.1 plasmids were transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

Techniques: Migration, Transfection, Cell Counting, Colony Assay, Incubation, Staining, Wound Healing Assay, Western Blot