Journal: Aging and Disease

Article Title: Mash1-dependent Notch Signaling Pathway Regulates GABAergic Neuron-Like Differentiation from Bone Marrow-Derived Mesenchymal Stem Cells

doi: 10.14336/AD.2016.1018

Figure Lengend Snippet: Primer sequences for Notch signaling and GABAergic neuron marker

Article Snippet: For RBPJ or Hes1 silencing, BMSCs which reached approximately 50% confluence in a 12-well plate were replaced with Polybrene (Santa Cruz Biotechnology, Inc., sc-134220, CA, USA) media mixture, followed by infection with RBPJ (Santa Cruz Biotechnology, Inc., sc-270318-v, CA, USA) or Hes1 (Santa Cruz Biotechnology, Inc., sc-270146-v, CA, USA) shRNA lentiviral particles (these primers were provided by the company) overnight (37°C, 5% CO 2 ).

Techniques: Sequencing

Journal: Aging and Disease

Article Title: Mash1-dependent Notch Signaling Pathway Regulates GABAergic Neuron-Like Differentiation from Bone Marrow-Derived Mesenchymal Stem Cells

doi: 10.14336/AD.2016.1018

Figure Lengend Snippet: A ) Protein bands of Notch signaling (RBPJ, Hes1 and Mash1) visualized through western blotting of BMSCs that were treated by DAPT (DAPT+BMSCs), or engineered by Hes1 shRNA (Hes1-BMSCs), pGC-FU-Mash1 plasmid (Mash1+BMSCs), RBPJ shRNA (RBPJ-BMSCs) and copGFP Control (Lv-con-BMSCs). B ) The protein level based on the ratio of Gauss Model Trace exhibited the Notch signaling (RBPJ, Hes1 and Mash1) changes in the DAPT+ BMSCs. C ) The protein level based on the ratio of Gauss Model Trace exhibited genetically engineered BMSCs. Error bars in bar graphs display standard deviation (SD). * P = 0.032, < 0.05, ** P = 0.0006, < 0.01.

Article Snippet: For RBPJ or Hes1 silencing, BMSCs which reached approximately 50% confluence in a 12-well plate were replaced with Polybrene (Santa Cruz Biotechnology, Inc., sc-134220, CA, USA) media mixture, followed by infection with RBPJ (Santa Cruz Biotechnology, Inc., sc-270318-v, CA, USA) or Hes1 (Santa Cruz Biotechnology, Inc., sc-270146-v, CA, USA) shRNA lentiviral particles (these primers were provided by the company) overnight (37°C, 5% CO 2 ).

Techniques: Western Blot, shRNA, Plasmid Preparation, Control, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: USP45-mediated deubiquitination of HIV-1 Tat regulates viral transcription and latency

doi: 10.3389/fmicb.2026.1656512

Figure Lengend Snippet: Comprehensive screening identifies DUBs that interact with HIV-1 Tat protein. (A) NanoBRET screening results of 120 DUB expression vectors for interaction with HIV-1 Tat. HEK293T cells were co-transfected with NanoLuc-fused Tat and HaloTag-fused DUBs. The top five candidates (CDKN2A, USP12, ATXN3L, USP45, and USP11) are highlighted. Data represent mean from two independent experiments. (B) Validation of Tat-DUB interactions by immunoprecipitation. HEK293T cells were co-transfected with HA-tagged Tat and the indicated DUB constructs. Cell lysates were immunoprecipitated with anti-HA antibody, and co-precipitated DUBs were detected by Western blotting.

Article Snippet: For stable knockdown experiments, cells were transduced with lentiviral vectors expressing shRNA targeting USP45 (Santa Cruz Biotechnology, #sc-76859-V) or control shRNA (#sc-108080), followed by puromycin selection (1 μg/ml) for 7 days.

Techniques: Expressing, Transfection, Biomarker Discovery, Immunoprecipitation, Construct, Western Blot

Journal: Frontiers in Microbiology

Article Title: USP45-mediated deubiquitination of HIV-1 Tat regulates viral transcription and latency

doi: 10.3389/fmicb.2026.1656512

Figure Lengend Snippet: USP45 mediates enzymatic activity-dependent deubiquitination of Tat protein. (A) Deubiquitinating activity of candidate DUBs toward Tat protein. HEK293T cells were co-transfected with HA-tagged Tat, HiBiT-tagged ubiquitin, and the indicated HaloTag-fused DUBs. Tat ubiquitination levels were assessed by immunoprecipitation with anti-HA antibody followed by Western blotting with HiBiT detection. Quantification shows relative HiBiT-ubiquitin levels normalized to empty vector control. (B) Catalytic activity requirement for USP45-mediated Tat deubiquitination. HEK293T cells were transfected with HA-tagged Tat, HiBiT-tagged ubiquitin, and either wild-type USP45 or catalytically inactive USP45 C199A mutant. Data represent mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For stable knockdown experiments, cells were transduced with lentiviral vectors expressing shRNA targeting USP45 (Santa Cruz Biotechnology, #sc-76859-V) or control shRNA (#sc-108080), followed by puromycin selection (1 μg/ml) for 7 days.

Techniques: Activity Assay, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Plasmid Preparation, Control, Mutagenesis

Journal: Frontiers in Microbiology

Article Title: USP45-mediated deubiquitination of HIV-1 Tat regulates viral transcription and latency

doi: 10.3389/fmicb.2026.1656512

Figure Lengend Snippet: USP45 suppresses Tat transcriptional activity and HIV-1 replication. (A) Effect of USP45 overexpression on HIV-1 LTR-driven transcription. HEK293T cells were co-transfected with HIV-1 LTR-luciferase reporter, Tat-HA, and increasing amounts of wild-type or catalytically inactive (C199A) USP45. Luciferase activity was measured 48 h post-transfection. *** p < 0.001. (B) USP45 reduces HIV-1 particle production. HEK293T cells were transfected with HIV-1 proviral DNA and increasing amounts of USP45 (wild-type or C199A). Viral production was assessed by p24 ELISA of culture supernatants at 48 h post-transfection. ** p < 0.01, *** p < 0.001. (C) USP45 overexpression decreases Tat protein levels. Western blot analysis of cells from (A) expressing USP45 (WT or C199A) and Tat-HA. (D-G) Effects of endogenous USP45 knockdown on HIV-1 replication. RT-qPCR analysis confirming approximately 60% USP45 knockdown efficiency using specific siRNA (D) . USP45 knockdown enhances HIV-1 LTR transcriptional activity (E) and viral particle production (F) compared to control siRNA. Long-term replication assay in Jurkat T cells with stable USP45 knockdown (shUSP45) shows enhanced HIV-1 replication (G) . Statistical comparisons were made against the control cells. * p < 0.05, ** p < 0.01.

Article Snippet: For stable knockdown experiments, cells were transduced with lentiviral vectors expressing shRNA targeting USP45 (Santa Cruz Biotechnology, #sc-76859-V) or control shRNA (#sc-108080), followed by puromycin selection (1 μg/ml) for 7 days.

Techniques: Activity Assay, Over Expression, Transfection, Luciferase, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Knockdown, Quantitative RT-PCR, Control

Journal: Frontiers in Microbiology

Article Title: USP45-mediated deubiquitination of HIV-1 Tat regulates viral transcription and latency

doi: 10.3389/fmicb.2026.1656512

Figure Lengend Snippet: USP45 specifically targets Tat lysine 19 residue for deubiquitination. (A) Mapping of USP45 target site on Tat protein. HEK293T cells were co-transfected with wild-type or lysine-to-alanine mutant Tat-HA constructs, HiBiT-tagged ubiquitin, and USP45. Tat ubiquitination was assessed by immunoprecipitation followed by HiBiT detection. Quantification shows relative ubiquitin levels normalized to the respective controls without USP45. (B) Functional importance of Tat K19 in transcriptional activity. HIV-1 LTR-luciferase reporter assay of cells expressing Tat (WT or K19A). ** p < 0.01, *** p < 0.001. (C) The K19A mutation reduces USP45-dependent enhancement of viral replication. Jurkat T cells with stable USP45 knockdown were infected with wild-type or K19A mutant HIV-1. p24 levels were measured by ELISA over 7 days post-infection. * p < 0.05.

Article Snippet: For stable knockdown experiments, cells were transduced with lentiviral vectors expressing shRNA targeting USP45 (Santa Cruz Biotechnology, #sc-76859-V) or control shRNA (#sc-108080), followed by puromycin selection (1 μg/ml) for 7 days.

Techniques: Residue, Transfection, Mutagenesis, Construct, Ubiquitin Proteomics, Immunoprecipitation, Functional Assay, Activity Assay, Luciferase, Reporter Assay, Expressing, Knockdown, Infection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: USP45-mediated deubiquitination of HIV-1 Tat regulates viral transcription and latency

doi: 10.3389/fmicb.2026.1656512

Figure Lengend Snippet: USP45 inhibits early stages of HIV-1 transcription and contributes to latency maintenance. (A) Schematic representation of HIV-1 transcriptional profiling assay. The HIV-1 proviral genome is shown with primer/probe locations for detecting five distinct transcriptional products: Read-through transcripts (gray) indicating transcriptional interference, TAR transcripts (red) representing transcriptional initiation, LongLTR transcripts (orange) indicating 5′ elongation, PolyA transcripts (blue) representing transcription completion, and Tat-Rev transcripts (purple) indicating splicing completion. (B) Transcriptional profiling of HIV-1 latency reversal in USP45 knockdown cells. J-Lat 8.4 cells with control or USP45 shRNA were treated with latency reversing agents (TNFα 10 ng/ml or SAHA 1 μM) or DMSO control. RNA was extracted and analyzed by RT-dPCR using stage-specific primer sets. Viral transcript levels were quantified by digital PCR and normalized to RNaseP gene expression as an internal control. Data represent mean ± SD from three independent experiments.

Article Snippet: For stable knockdown experiments, cells were transduced with lentiviral vectors expressing shRNA targeting USP45 (Santa Cruz Biotechnology, #sc-76859-V) or control shRNA (#sc-108080), followed by puromycin selection (1 μg/ml) for 7 days.

Techniques: Knockdown, Control, shRNA, Digital PCR, Gene Expression

Journal: Frontiers in Microbiology

Article Title: USP45-mediated deubiquitination of HIV-1 Tat regulates viral transcription and latency

doi: 10.3389/fmicb.2026.1656512

Figure Lengend Snippet: USP45 is induced by interferon treatment. (A) USP45 mRNA and (B) protein expression levels in HEK293T and Jurkat T cells treated with type I (IFN-α, IFN-β), type II (IFN-γ), or type III (IFN-λ1) interferons for 24 h. mRNA levels were analyzed by qPCR and normalized to untreated controls. Protein expression was analyzed by immunoblotting with tubulin as loading control. Data represent mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: For stable knockdown experiments, cells were transduced with lentiviral vectors expressing shRNA targeting USP45 (Santa Cruz Biotechnology, #sc-76859-V) or control shRNA (#sc-108080), followed by puromycin selection (1 μg/ml) for 7 days.

Techniques: Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: EGFR endocytosis is a novel therapeutic target in lung cancer with wild-type EGFR

doi:

Figure Lengend Snippet: (A) Difference in endosomal localization of EGF-induced EGFR between H358 and H1703 cells was profiled by immunofuorescence staining with the indicated antibodies. The immunofluorescence images were obtained using a fluorescence microscope at a magnification of 400×. Each white arrow indicates co-localization (yellow). (B) Effect of Rab25 expression on EGFR endocytosis was examined in siRab25 transfected H358 cells by flow cytometry. After siRab25 or scrambled (Scr) transfection, the cells were incubated with gefitinib (10 μM) and the percentage of internalized Alexa Fluor 488-conjugated EGF (500 ng/ml) was analyzed. Each bar represents mean values acquired from three experiments with standard error (SE). * p < 0.05. (C) Correlation between Rab25 expression and gefitinib response was determined by comparative analysis. Expression of Rab25 mRNA was evaluated in eight lung cancer cell lines with wtEGFR using quantitative RT-PCR. Relative Rab25 expression values were normalized to values of GAPDH. IC 50 of each cell was obtained from MTT assay results. The P value represents statistical comparisons between Rab25 expression and gefitinib response. Red: gefitinib-sensitive cell lines. Blue: gefitinib-insensitive cell lines.

Article Snippet: After replacing the culture medium with a Polybrene/media mixture, the cells were incubated with shScrambled control (Scr) and Rab25 shRNA lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA) overnight.

Techniques: Staining, Immunofluorescence, Fluorescence, Microscopy, Expressing, Transfection, Flow Cytometry, Incubation, Quantitative RT-PCR, MTT Assay

Journal: Oncotarget

Article Title: EGFR endocytosis is a novel therapeutic target in lung cancer with wild-type EGFR

doi:

Figure Lengend Snippet: After siRab25 or scrambled (Scr) transfection, H358 cells were treated with the indicated concentrations of gefitinib. (A) Changed gefitinib response based on Rab25 expression status was monitored by light microscopy. Cell images were captured at a magnification of 100×. Cell viability (B) and IC 50 (C) in siRab25 or Scr transfected cells were determined after gefitinib treatment using MTT assay. Each data point represents mean results of six independent determinations with standard error. * p < 0.05. (D) Redistributed percentages of the cell cycle caused by Rab25 expression status were analyzed after gefitinib treatment using flow cytometry. Data represents mean results acquired from three independent experiments.

Article Snippet: After replacing the culture medium with a Polybrene/media mixture, the cells were incubated with shScrambled control (Scr) and Rab25 shRNA lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA) overnight.

Techniques: Transfection, Expressing, Light Microscopy, MTT Assay, Flow Cytometry