Journal: Nature cell biology

Article Title: Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver

doi: 10.1038/ncb3169

Figure Lengend Snippet: (a) Representative images and quantification of pre-metastatic niche markers in livers educated with PAN02 exosomes (Exo), PAN02shCTL exosomes (shCTLexo), PAN02shMIF exosomes (shMIFexo and shMIF(2)exo) or PBS control (CTL). Immunofluorescence analysis shows frequency of TGFβ-expressing F4/80+ cells, αSMA and FN expression as well as F4/80+ cell frequency. Inset shows TGFβ+/F4/80+ cell; n = 3 (CTL F4/80), n = 4 (CTL TGFβ, αSMA, FN; Exo TGFβ; all shCTL; shMIF TGFβ; all shMIF(2), n = 6 (Exo F4/80; shMIF F4/80), n = 7 (Exo αSMA, FN; shMIF αSMA, FN) mice pooled from two experiments. ***P < 0.001, **P < 0.001 by ANOVA. Scale bars, 100μm. (b) Evaluation of liver metastasis by liver weight (grams) in tumor-free mice (CTL), mice injected intra-splenically with PAN02 cells either pre-educated with PBS (TU), with PAN02 exosomes (Exo+TU), with PAN02 exosomes in combination with A83-01 (Exo+A83-01+TU), or with PAN02shMIF exosomes (shMIFexo+TU); n = 4 (Exo+A83-01+TU), n = 5 (CTL, TU, Exo+TU, and shMIFexo+TU) mice pooled from two experiments. **P < 0.01, *P < 0.05 by ANOVA. Scale bar, 1cm. (c) Evaluation of liver metastasis in mice injected intra-splenically with PAN02 cells either pre-educated with PBS (TU), with exosomes isolated from PAN02 cells infected with control shRNA (shCTLexo+TU) or shMIF(2) (shMIF(2)exo+TU) lentiviral vectors; n = 4 (shCTLexo+TU), n = 5 (shMIF(2)exo+TU) and n = 6 (TU) mice from one experiment. ***P < 0.01 by ANOVA. Scale bar, 1cm. (d) Enzyme-linked immune assay (ELISA) reveals increased levels of MIF (picogram per 108 exosomes) in exosomes isolated from patients with pancreatic ductal adenocarcinoma (PDAC) with progression of disease post-diagnosis (POD) compared to PDAC patients with no evidence of disease 5 years post-diagnosis (NED) and to healthy controls (CTL), but not PDAC patients with liver metastasis (LM); n = 10 (NED), n = 12 (POD), n = 15 (CTL) and n = 18 (LM) patients. All patient samples were analyzed once as part of three independent ELISA assays. **P < 0.01 by ANOVA. All data are represented as mean±s.e.m

Article Snippet: MIF knockdown PAN02 cells were generated by using MIF shRNA lentiviral particle infection, generating the PAN02 variants shMIF (sc-37138-V, Santa Cruz) and shMIF(2) (iV042354, abmGood).

Techniques: Control, Immunofluorescence, Expressing, Injection, Isolation, Infection, shRNA, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

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: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung

doi: 10.1152/ajplung.00324.2017

Figure Lengend Snippet: Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) defines genome-wide transcriptional regulation by pSmad1 protein upon growth differentiation factor 15 (GDF15) in human airway epithelial cells. Normal human tracheobronchial epithelial cells in submerged culture were treated with 0.1% BSA-HCl (control) or recombinant human GDF15 (25 ng/ml) for 2 h to perform the phosphorylated-Smad1 (pSmad1) ChIP-seq analysis.

Article Snippet: Lentiviruses encoding human Smad1 shRNA (sc-29483-v, shSmad1) or control shRNA (sc-108080, shControl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Chromatin Immunoprecipitation, Next-Generation Sequencing, ChIP-sequencing, Genome Wide, Control, Recombinant

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung

doi: 10.1152/ajplung.00324.2017

Figure Lengend Snippet: Overexpressing human growth differentiation factor 15 (GDF15) enhances activation of Smad1 and interferon regulatory factor 7 (IRF7) in mouse airway epithelial cells. Primary tracheal epithelial cells from naïve human GDF15 transgenic (hGDF15 Tg+) mice or wild-type (WT) littermates were grown at air-liquid interface and infected with HRV-1B [multiplicity of infection (MOI) 1] or PBS (control) for 24 h. A, left: quantitative Western blot densitometry data of phosphorylated-Smad1 (pSmad1) and total Smad1 protein. A, right: representative Western blot pictures; B, left: quantitative Western blot densitometry data of IRF7 and β-actin protein (loading control). B, right: representative Western blot pictures. Data are presented as means ± SE from three independent experiments. *P < 0.05, **P < 0.01, compared with PBS controls or HRV-infected WT cells.

Article Snippet: Lentiviruses encoding human Smad1 shRNA (sc-29483-v, shSmad1) or control shRNA (sc-108080, shControl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Transgenic Assay, Infection, Control, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung

doi: 10.1152/ajplung.00324.2017

Figure Lengend Snippet: Smad1 cooperates with interferon regulatory factor 7 (IRF7) in response to HRV infection via growth differentiation factor 15 (GDF15) signaling in human airway epithelial cells. A: normal human bronchial epithelial cells transduced with lentiviruses encoding human GDF15 shRNA (shGDF15) or control shRNA (shControl) were grown at air-liquid interface and infected with HRV-16 [multiplicity of infection (MOI) 1] or PBS (control) for 24 h. A, top: quantitative Western blot densitometry data of IRF7 and β-actin protein (loading control). A, bottom: representative Western blot pictures. B, top: normal human tracheobronchial epithelial cells transduced with lentiviruses encoding human Smad1 shRNA (shSmad1) or control shRNA (shControl) were grown at air-liquid interface for 28 days to examine Smad1 protein expression. B, bottom: quantitative Western blot densitometry data of Smad1 and GAPDH protein (loading control). C: normal human tracheobronchial epithelial cells transduced with lentiviruses encoding human Smad1 shRNA (shSmad1) or control shRNA (shControl) were grown at air-liquid interface, pretreated with 0.1% BSA-HCl (control) or recombinant human GDF15 (25 ng/ml) for 72 h, and infected with HRV-16 (MOI 1) or PBS for 24 h. C, top: quantitative Western blot densitometry data of IRF7 and β-actin protein (loading control). C, bottom: representative Western blot pictures. Data are presented as means ± SE from 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with PBS controls or shControl cells; NS, not significant.

Article Snippet: Lentiviruses encoding human Smad1 shRNA (sc-29483-v, shSmad1) or control shRNA (sc-108080, shControl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Infection, Transduction, shRNA, Control, Western Blot, Expressing, Recombinant

Journal: The Journal of Neuroscience

Article Title: Interaction of nNOS with PSD-95 Negatively Controls Regenerative Repair after Stroke

doi: 10.1523/JNEUROSCI.1305-14.2014

Figure Lengend Snippet: HDAC2 in NSCs is a mediator for the role of neuronal nNOS–PSD-95 association in regulating the fate of NSCs. A, B, Immunoblots showing HDAC2 levels (n = 4; A) and bar graph showing the percentage of newborn neurons (n = 3; B) in the NSCs infected by LV-HDAC2-shRNA, LV-control-shRNA, AD-HDAC2-Flag, or AD-Flag. C, Bar graph showing the percentage of newborn neurons in the DG of rats infected by AD-HDAC2-Flag or AD-Flag (n = 9). These rats were subjected to MCAO at 24 h after virus infection and were killed for BrdU/DCX staining at day 14 after MCAO administration. D–F, Bar graphs showing the percentage of newborn neurons (D), the percentage of neurons with multineurites (E), and total neurite length per newborn neuron with multineurites (F) in the NSCs treated with DETA/NONOate (50 μm) with or without NO scavenger C-PTIO (10 μm) for 24 h at day 2 after differentiation. The cultures were stained 4–5 d after treatment. (n = 3). G, Nitrosylation of total proteins or HDAC2 in NSCs. NSCs were treated with 50 μm DETA/NONOate for the indicated times (G1), with 50 μm GSNO for 4 h (G2), or with 50 μm DETA/NONOate for 4 h (G3) at day 2 after differentiation (n = 4). PC, Positive control, in which the lysate of NSCs was treated with GSNO (200 μm) for 30 min; BSA, biotin-switch assay. H, Immunoblots showing HDAC2 levels in the NSCs treated with 50 μm DETA/NONOate for the indicated times at day 2 after differentiation. (n = 3–5). I, Bar graphs showing HDAC2 activity in the NSCs treated with 50 μm DETA/NONOate for the indicated times (left) or by 50 μm DETA/NONOate with or without 10 μm NO scavenger C-PTIO for 4 h (right) at day 2 after differentiation. n = 3–5. J, K, HDAC2 levels (J; n = 5) and enzymatic activity (K; n = 4) in the solo-cultured or neuron-cocultured NSCs with or without C-PTIO. All cultures were exposed to OGD for 2 h at day 2 after NSC differentiation, and the NSCs were collected 6 h after OGD finish. To measure HDAC2 in NSCs selectively, coculture was performed with neurons in membrane inserts (1 μm pore size) and NSCs in wells. C-PTIO (10 μm) or vehicle was added to the medium of NSCs in wells at the start of OGD exposure. L, M, The percentage of newborn neurons (L) and the percentage of newborn neurons with multineurites (M) in the cocultures of LV-control-shRNA- or LV-HDAC2-shRNA-infected NSCs with GFP+ neurons. The cocultures were subjected to OGD for 2 h at day 2 after differentiation and stained at days 4∼5 after OGD (n = 4). N, O, Bar graphs showing the percentage of newborn neurons (N) and the percentage of newborn neurons with multineurites (O) in the cocultures of AD-HDAC2-flag- or AD-flag-infected NSCs with GFP+ neurons. The cocultures were subjected to OGD for 2 h and treated with 10 μm ZL006 for 26 h beginning at OGD administration, and were stained at days 4∼5 after OGD administration. n = 5. Data are the mean ± SEM. *p < 0.05, **p < 0.01, two-tailed t test for C, ANOVA for others.

Article Snippet: HDAC2 shRNA (m) lentiviral particles (LV-HDAC2 shRNA, Santa Cruz Biotechnology) are recommended for the inhibition of HDAC2 expression in mouse cells.

Techniques: Western Blot, Infection, shRNA, Control, Virus, Staining, Positive Control, Biotin Switch Assay, Activity Assay, Cell Culture, Membrane, Pore Size, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Interaction of nNOS with PSD-95 Negatively Controls Regenerative Repair after Stroke

doi: 10.1523/JNEUROSCI.1305-14.2014

Figure Lengend Snippet: Association of nNOS–PSD-95 in neurons and consequent NO production may negatively regulate NSC fate via HDAC2-mediated histone deacetylation and NeuroD downregulation. A, B, Immunoblots showing acetyl-H4 (A) and NeuroD (B) levels in the NSCs treated by 50 μm DETA/NONOate with or without 10 μm C-PTIO for 8 h at day 2 after differentiation (n = 4). C, D, Immunoblots showing acetyl-H4 (C) and NeuroD (D) levels in the NSCs treated by 50 μm DETA/NONOate with or without 2 nm TSA (an HDAC inhibitor that was added 30 min before DETA/NONOate) for 8 h at day 2 after differentiation (n = 4). E, F, Immunoblots showing acetyl-H4 (E) and NeuroD (F) levels in the NSCs treated with 50 μm DETA/NONOate with or without LV-HDAC2-shRNA infection for 8 h at day 2 after differentiation (n = 4). DETA/NONOate was added at day 6 after LV-HDAC2-shRNA or LV-control-shRNA infection. G, H, Immunoblots (G) and immunofluorescence (H) showing acetylation of histone H4 in the NSCs cocultured with neurons after OGD. Coculture was performed with neurons in membrane inserts (1 μm pore size) and NSCs in wells (n = 4). Data are the mean ± SEM. *p < 0.05, **p < 0.01, ANOVA.

Article Snippet: HDAC2 shRNA (m) lentiviral particles (LV-HDAC2 shRNA, Santa Cruz Biotechnology) are recommended for the inhibition of HDAC2 expression in mouse cells.

Techniques: Western Blot, shRNA, Infection, Control, Immunofluorescence, Membrane, Pore Size

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