Journal: Journal of Thoracic Disease

Article Title: Yifei Sanjie Formula in improving the response to PD-1 blockade by lung cancer through the attenuation of the USP7-NR1H4-bile acid metabolism axis

doi: 10.21037/jtd-2026-1-0326

Figure Lengend Snippet: USP7 facilitated the migration and proliferation ability of LLC cells in vitro and in vivo . (A) Western blot analysis of USP7-knockdown efficiency for three shRNAs. (B,C) CCK-8 proliferation and colony-formation assays were used to detect the proliferation ability in USP7-knockdown LLC cells (Giemsa staining). (D) Cell-cycle analysis revealed that knocking down USP7 expression in LLC cells increased the percentage of cells in G1 phase. (E,F) USP7 knockdown markedly attenuated cell migration in LLC cells as measured by Transwell migration and wound-healing assays. Cells on the transwell membrane were stained with Giemsa. Images were taken with a microscope at 100×. (G) Representative images of the gross tumors are shown. Gross view of the xenograft tumors in C57BL/6 mice. Tumors derived from LLC cells expressing sh-USP7 grew significantly slower than did those from cells with sh-Ctrl. Cells were injected into the hindlimbs of C57BL/6 mice (n=6). (H,I) Final tumor weights and body weight were measured in each group. (J) Tumor growth curves were measured during the growth of the tumors. (K) The xenograft tumors sections were stained with H&E. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; Ctrl, control; H&E, hematoxylin and eosin; LLC, Lewis lung carcinoma; OD, optical density; shRNA, short hairpin RNA; USP7, ubiquitin-specific protease 7.

Article Snippet: USP7-knockdown cells were treated with MG132 (cat. no. HY-13259, MedChemExpress), a proteasome inhibitor.

Techniques: Migration, In Vitro, In Vivo, Western Blot, Knockdown, CCK-8 Assay, Staining, Cell Cycle Assay, Expressing, Membrane, Microscopy, Derivative Assay, Injection, Cell Counting, Control, shRNA, Ubiquitin Proteomics

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 1. Hypoxia regulates GCM1 degradation. A, both GCM1 transcript and protein levels are decreased in placental cells under hypoxia. Total RNA was purified from BeWo, BeWo31, and JAR cells under normoxia (white bars) or hypoxia (black bars) for 48 h. One g of RNA was converted into the first strand cDNA using oligo(dT)20 as primer, followed by quantitative real time PCR. Mean values and S.D. of the ratio of GCM1 to -actin RNA copy number are shown (left). Cell protein extracts from BeWo cells under normoxia or hypoxia for 48 h were immunoblotted with GCM1 or -tubulin antibody (right). B, hypoxia regulates GCM1 expression at the post-translational level. BeWo31 cells were cultured under hypoxia for 48 h or exposed to 250 M CoCl2 for 36 or 48 h. Cells were then harvested for immunoblotting with HA or -tubulin antibody (left) or for RT-PCR analysis of the transcript levels of HA-GCM1 and glyceraldehyde-3-phosphate dehydrogenase. C, the effect of hypoxia on GCM1 protein level is reversible. BeWo31 cells were mock-treated or pretreated with 250 M CoCl2 for 24 h. The medium was then replaced with fresh medium without CoCl2, and culture was continued for the indicated period of time before harvesting the cells for immunoblotting with HA or -actin antibody. D, proteasome is involved in the decreased GCM1 protein level induced by hypoxia. BeWo31 cells were mock-treated or treated with 250 M CoCl2 in the presence or absence of 40 M MG132 for 24 h. Cells were then harvested for immunoblotting with HA or -actin antibody.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Purification, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 3. GSK-3 controls GCM1 ubiquitination and degradation. A, GCM1 ubiquitination is regulated by GSK-3. 293T cells were transfected with 1 g of pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid. At 24 h post-transfection, cells were treated with 40 M MG132 for an additional 4 h and thensubjecttoubiquitinationanalysisbyimmunoprecipitation(IP)withFLAGmAbandimmunoblotting(IB)withHAmAb(left).Inaseparateexperiment,293T cellsweretransfectedwith1gofpGCM1-FLAGandpHA-Ubplus10nMGL2orGSK-3siRNAandincubatedasaboveforubiquitinationanalysis.B,thehalf-life of GCM1 is prolonged by inhibition of GSK-3. 293T cells were transfected with 0.35 g of pGCM1-FLAG. At 24 h post-transfection, cells were pretreated with or without 50 mM LiCl for 2 h before incubation with 75 M cycloheximide for the indicated period of time. In a separate experiment, 293T cells were transfected with0.35gofpGCM1-FLAGplus10nMGL2orGSK-3siRNA.At24hpost-transfection,cellswereincubatedwith75Mcycloheximidefortheindicatedperiod of time. Cells were then harvested for immunoblotting with FLAG or -tubulin antibody.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Inhibition, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 4. Identification of GSK-3 phosphorylation sites and a C-terminal destruction motif in GCM1. A, Ser322 and Ser326 are required for GCM1 ubiquitination. 293T cells were transfected with 1 g of pHA-Ub and wild-type or mutant pGCM1-FLAG, treated with MG132, and subject to ubiquitination analysis as described in the legend to Fig. 3A. B, Ser322 and Ser326 are involved in regulation of GCM1 stability. 293T cells were transfected with the indicated pGCM1-FLAG expression plasmid for protein stability analysis as described in the legend to Fig. 3B. C and D, FBW2 interacts with the C-terminal TpSWPCpS (where pS represents phosphoserine) destruction motif in GCM1. GST- or GST-FBW2-loaded glutathione beads were incubated with 100 g of the cell lysate prepared from 293T cells transfected with wild-type or mutant pGCM1-FLAG expression plasmids for pull-down analysis, followed by immunoblotting with FLAG mAb. Biotinylated unmodified peptides or phosphopeptides covering amino acids 313–333 were incubated with 100 g of cell lysate prepared from 293T cells transfected with pFBW2-Myc, pTrcp-Myc, or pSKP2-Myc for pull-down analysis, followed by immunoblotting with Myc mAb. IP, immunoprecipi- tation; IB, immunoblot.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 5. Hypoxia induces Ser322 phosphorylation in GCM1 in vivo. A, GSK-3 phosphorylates Ser322 under hypoxia. 293T cells were transfected with pHA-GCM1 plus GL2 or GSK-3 siRNA. The transfected cells were subjected to hypoxic conditions for 24 h in the presence of MG132, followed by immunoprecipitation (IP) with HA mAb and immunoblotting (IB) with Ser(P)322-GCM1 Ab. B, Ser322 phosphorylation in placental cells under hypoxia. BeWo31 cells were cultured under normoxia or hypoxia for 48 h and mock-treated or treated with the indicated combination of MG132 and LiCl, followed by immunoprecipitation with HA mAb and immunoblot- ting with HA, Ser(P)322-GCM1, or Ser(P)326-GCM1 antibody. C, GCM1 degradation under placental hypoxia contributes to the pathogenesis of preeclampsia.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Phospho-proteomics, In Vivo, Transfection, Immunoprecipitation, Western Blot, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

doi: 10.1083/jcb.201506048

Figure Lengend Snippet: TRIM3 polyubiquitylates ACTG1. (A–D) TPA stimulation induces ACTG1 polyubiquitylation by TRIM3 in HEK293 cells. HEK293 cells were transfected with TRIM3 or ΔRBCC-TRIM3. After incubation for 4 h in the presence of MG132, cells were lysed and ACTG1 was immunoprecipitated from the lysates, resolved on SDS-PAGE, and immunoblotted. Blots were first stained for polyubiquitin (A) and then stripped and restained for ACTG1 (B). Under these basal conditions, only unmodified ACTG1 was detected. However, when cells were incubated in the presence of MG132 and TPA, a significant increase in high-molecular-weight polyubiquitylated forms of ACTG1 (indicated with *) was observed specifically in TRIM3-transfected cells (C–E; means ± SEM, two-tailed t test, *, P < 0.05, n = 3 cultures per condition). (F) TPA induces ACTG1 polyubiquitylation by TRIM3 in hippocampal neurons. Cultured hippocampal neurons from wild-type and Trim3 −/− mice were treated with MG132 and TPA for 4 h. Lysates were immunoblotted and stained for ACTG1. TPA induced the appearance of multiple high molecular weight (50–100 kD) bands (indicated with *) consistent with polyubiquitylation and specifically in neurons from wild-type mice and not from Trim3 −/− mice. (G) Actg1 mRNA is present in TRIM3/PURA containing mRNP granules. mRNP granules were immunoprecipitated from hippocampal lysates with antibodies against TRIM3 or PURA. mRNA was isolated from immunoprecipitates, reverse transcribed into cDNA, and used for real-time quantitative PCR. Actg1 and Actb mRNA was detected at equal levels in all precipitates, whereas the negative control mRNA Slc1a3 was ∼10-fold lower detected (means ± SEM, n = 3 immunoprecipitations per condition).

Article Snippet: After 14 d in culture, neurons were either lysed in SDS loading buffer directly or first incubated with 20 μM MG132 (Tocris Bioscience) and 500 nM TPA (Sigma-Aldrich) for 4 h and then lysed.

Techniques: Transfection, Incubation, Immunoprecipitation, SDS Page, Staining, High Molecular Weight, Two Tailed Test, Cell Culture, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: δ-opioid receptor (DOR)-expressing HEK293 cells and HEK293 cells stained with anti-DOR antibody and followed with FITC-conjugated anti-mouse IgG ( A ). Volcano plot of identified differentially expressed genes (DEGs) in DOR-HEK293 cells after 1 h treatment with CM-10 ( B ). Red boxes show changed gene expression over 1.4-time higher and less than 0.71 with p -value predicted by EdgeR of less than 0.05.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Expressing, Staining, Gene Expression

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: Predicted DOR agonistic signaling networks in DOR-HEK293 cells after 1 h treatment with CM-10. Fourteen genes with suggested involvement in cAMP-dependent protein kinases, transcriptional regulators in response to cAMP, circadian rhythm, stress and depression are shown in and were applied for network analysis by STRING. Red: circadian rhythm, Green: regulation of transcription of Notch receptor target, Yellow: PKA activation in glucagon signalling.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Activation Assay