Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Cell cycle analysis of VP40 clones. 293T, V2CL, and V2CH cells were seeded in a 96-well plate at 5 × 10 5 cells in 100 μL of fresh media. Cells were blocked at G 0 (starvation; 0.1% fetal bovine serum DMEM), G 1 /S (20 mM of hydroxyurea), and G 2 /M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour, and subsequent treatment with 50 ng/mL of nocodazole) for 2 days. Blocked cells were then imaged (A) and assayed for cell viability with CellTiter-Glo (B). The same experiment was repeated; however, cells were allowed to incubate for 5 days after blocking. Cells were subsequently imaged (C) and assayed for cell viability via CellTiter-Glo (D). Statistical analysis by Student’s 2-tailed t test compares V2CL and V2CH cell cycle-blocked groups with corresponding 293T groups (†, P < .05; ††, P < .01; †††, P < .001). Additional Student’s 2-tailed t test compares cell cycle-blocked groups with controls of their own cell type (**, P < .01; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Cell Cycle Assay, Clone Assay, Blocking Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Effect of nuclear VP40 on cyclin and cdk regulation and activity. (A) Log phase 293T, V2CL, V2CI, and V2CH cells were harvested, lysed, and subjected to SDS/PAGE for western blot analysis of cyclin D1 (CycD1), cyclin E (CycE), cyclin A (CycA), cyclin B1 (CycB1), cdk4, cdk6, cdk2, cdk1, and actin levels. (B) Five hundred micrograms of 293T or V2CH whole-cell extracts were used for IP with 10 µg of either normal rabbit immunoglobulin G (IgG) or α-CycD1. IPed material was incubated with 50 µL 30% of Protein A/G for 2 hours, followed by two 1× PBS washes and 1 kinase buffer wash. Pellets were resuspended in kinase buffer, and 15 µL samples were incubated with 50 µg of either no peptide, 780S, or 780A peptide, along with 2 µL of [γ- 32 P] ATP. Some samples were also treated with Fascaplysin ([Fascap.] 1 µM). All samples incubated for 1 hour, followed by dotting onto Whatman glass microfiber filters and drying for 30 minutes. Filters were incubated in 1× TE buffer with gentle agitation for 2 days, dried, and then quantified with a scintillation counter. Background levels of CycD1 kinase activity (α-CycD1 IP with 780A peptide substrate) are indicated by black bar. (C) Log phase 293T and V2CH cells were harvested for separation of cytoplasmic (Cyt) and nuclear (Nuc) compartments with the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific). Western blot analysis for levels of VP40, CHMP6, HDAC1, and Actin was performed. (D) Log phase 293T and V2CH cells (3 × 10 6 ) were harvested and cross-linked with 1% formaldehyde for 1 hour followed by quenching with 1.25 glycine (9:1 cell suspension/glycine). Samples were sonicated, and 100 µL of each sample (corresponding to approximately 5 × 10 5 cells) was used for IP with 1 µg of anti-Pol II, α-p300, or α-VP40 at 4°C overnight. The next day, Protein A/G (30% slurry) was added and incubated for 2 hours at 4°C. Complexes were washed once with each TNE300 + 0.1% NP40, TNE150 + 0.1% NP40, TNE50 + 0.1% NP40, and IP wash buffer before the addition of proteinase K (800 units/mL). Samples were incubated for 15 minutes at 65°C, reversing solution was added, and samples were incubated for an additional 90 minutes at 65°C. DNA was purified, and qPCR was performed with 2 μL of undiluted DNA with primers for the CycD1 promoter region (spanning −180 to +238 from the messenger RNA start site at +1). The absolute quantification of the samples was determined based on the cycle threshold value relative to the standard curve generated from serial dilutions of DNA from 293T cells. Data are presented as percentage (%) of the input (DNA purified from sonicated samples before IP with specific antibodies). Student’s 2-tailed t test compares V2CH ChIPed DNA with corresponding 293T ChIP samples (**, P < .01).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Activity Assay, SDS Page, Western Blot, Incubation, Sonication, Purification, Generated

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Alteration of cell viability by VP40 in multiple cell types. Cells including (A) U937, HeLa, and (B) 3 peripheral blood mononuclear cells (PBMCs) log-phase cultures (~1.65 × 10 5 cells) were transfected with attractene and 1.5 µg of cytomegalovirus-VP40 plasmid. The PBMCs received a 1-time treatment of 50 IU/mL of interleukin-2 the day before transfection. Control cells received attractene treatment alone. Cell viability was assayed 3 days post-transfection. Statistical analysis by Student’s 2-tailed t test compares control cells with transfected cells (*, P < .05; **, P < .01).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Transfection, Plasmid Preparation

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Differential biogenesis of exosomes at different phases of the cell cycle. 293T and V2CL cells were blocked at G 0 (starvation; 0.1% fetal bovine serum DMEM), G 1 /S (20 mM of hydroxyurea), and G 2 /M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour and subsequent treatment with 50 ng/mL of nocodazole) for 5 days. Control (unsynchronized) cells were also incubated for 5 days. Black arrows point to bands of noticeable difference between V2CL and 293T cells. (A) Blocked cells and controls were harvested, washed twice in 1× PBS, and lysed. Samples were run on a 4–20% Tris-glycine gel and analyzed by western blot for the presence of ESCRT pathway proteins (VPS4, EAP45, TSG101, CHMP6, and EAP20), exosomal markers (Alix and CD63), and Actin. (B) Cell-free supernatants from blocked cells were harvested and passed through a 0.22-µm filter. One milliliter of filtered supernatant was incubated with 30 μL of NT80/82 particles overnight at 4°C. The next day, the NT pellet was washed once in 1× PBS and resuspended in 10 μL Laemmli buffer, followed by SDS/PAGE and western blot analysis for VP40 protein, exosomal markers CD63 and Alix, and Actin. (C) One milliliter of filtered supernatant from blocked and control cells was incubated with 30 μL of NT80/82 particles overnight at 4°C. The next day, the NT pellets were isolated, washed, and subjected to AChE assay for quantification of exosomes. Statistical analysis was completed by Student’s 2-tailed t test (*, P < .05; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Incubation, Western Blot, SDS Page, Isolation, AChE Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Extracellular vesicles released by VP40-producing cells at different phases of the cell cycle. 293T, V2CL, and V2CH cells were blocked at G 0 (starvation; 0.1% fetal bovine serum DMEM), G 1 /S (20 mM of hydroxyurea), and G 2 /M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour and subsequent treatment with 50 ng/mL of nocodazole) for 5 days. Supernatants were harvested, filtered (0.22 μm), and analyzed by ZetaView for size (peak [mode] diameter) (A) and concentration of particles (B). Statistical analysis by Student’s 2-tailed t test compares V2CL and V2CH cell cycle-blocked groups with corresponding 293T groups (†, P < .05; ††, P < .01; †††, P < .001). Additional Student’s 2-tailed t test compares cell cycle-blocked groups with controls of their own cell type (*, P < .05; **, P < .01; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Concentration Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Iodixanol gradient separation of extracellular vesicles (EVs) from 293T and VP40-producing cells. 293T and V2CI cells were grown in exosome-free media for 5 days, followed by harvesting of the supernatant and incubation with ExoMAX (1:1 reagent/filtered supernatant) reagent overnight at 4°C. The EVs were pelleted, resuspended in 300 µL of sterile 1× PBS, and loaded onto a 6–18% iodixanol density gradient (1.2% increments). Samples were ultracentrifuged for 90 minutes at 100000 × g , followed by harvesting and isolation of each fraction, and incubation with 30 µL of NT80/82 particles overnight at 4°C. The NT pellets were washed in 1× PBS, resuspended in 12 µL of Laemmli buffer, and loaded onto a 4–20% Tris-glycine gel. Western blot of 293T (A) and V2CI (B) fractions were analyzed for levels of VP40, CD63, CD81, CD9, and Actin. Major groups of EVs or exosome type are indicated by black boxes.

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Incubation, Isolation, Western Blot

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: The presence of VP40 in exosomes in in vitro and in vivo EBOV-infected cells. (A) HUVECs were cultured and infected with EBOV (MOI of 1) and incubated for 3 days under BSL-4 containment. Two milliliters supernatant were harvested, passed through a 0.22-µm filter, and incubated with ExoMAX (1:1 reagent/filtered supernatant) reagent overnight at 4°C. EVs were pelleted, resuspended in 0.5 mL 1× PBS, and loaded on qEV columns. Fraction numbers 7–10 (0.5 mL each) were collected and separately incubated with 30 μL NT80/82 at room temperature for 1 hour. The EV-bound NTs were washed with 1× PBS, followed by resuspension in 10 μL 2× NuPAGE LDS sample buffer, heating at 95°C for 10 minutes, and loading onto a 4–12% Tris-glycine gel for subsequent western blot analysis for VP40, GP, NP, and Actin levels. Negative control (Null) samples consisted of purified exosomes from uninfected HUVECs. (B) Gamma-irradiated and inactivated NHP (rhesus monkey) serum samples were obtained. NHP 1: day 0 prebleed sample. NHP 2: pool of day 4 and day 5 postinfection (pi); NHP 2 died on day 7 post-EBOV infection. NHP 3: pool of day 8–11 pi; NHP 3 died on day 12 post-EBOV infection. One hundred microliters of serum were diluted with 400 μL sterile 1× PBS and filtered (0.22 µm). Twenty-five microliters NT80/82 particles were incubated with the filtered samples at 4°C overnight. The next day, NT pellets were washed once in 1× PBS, resuspended in 12 μL Laemmli buffer, run on 4–20% SDS/PAGE, and analyzed by western blot for VP40 protein and exosomal markers CD81 and CD9.

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: In Vitro, In Vivo, Infection, Cell Culture, Incubation, Western Blot, Negative Control, Purification, Irradiation, SDS Page

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Induction of recipient T-cell death by purified VP40 EVs. (A) 293T and V2CH cells were grown in exosome-free media for 5 days, followed by harvesting of cell-free supernatants and filtration through 0.22 μm. Supernatants were then spun at 100000 × g for 90 minutes to pellet EVs, followed by resuspension in sterile 1× PBS. Concentrations of resulting ultracentrifuged EVs were determined with ZetaView analysis, followed by treatment of CEM cells with increasing concentrations of EVs (10000, 25000, or 75000 particles/cell) from V2CH cell type. Controls included CEM cells that were left untreated and CEM cells that received a treatment of the highest concentration of 293T cells (75000 particles/cell). Cells were incubated for 3 days followed by analysis of cell viability by CellTiter-Glo. (B) 293T and V2CI cells were grown in exosome-free media for 5 days. Cell-free supernatants were harvested and incubated with equal volumes of ExoMAX overnight at 4°C. The EVs were pelleted, resuspended in 400 µL sterile 1× PBS, and loaded onto a 6–18% iodixanol density gradient (1.2% increments). Samples were ultracentrifuged for 90 minutes at 100000 × g , followed by harvesting and isolation of each fraction. Select fractions (13.2 + 14.4 and 16.8 + 18.0) were pooled and subjected to a second ultracentrifuge spin for 90 minutes at 100000 × g diluted in 1× PBS to pellet the EVs away from residual iodixanol. Resulting EV pellets were resuspended in 100 µL sterile 1× PBS and used to treat recipient CEM cells at a concentration of 10000 particles per cell (concentrations determined by ZetaView analysis). Cells were incubated for 5 days followed by analysis of viability by CellTiter-Glo assay. Statistical analysis by Student’s 2-tailed t test compares groups treated with EVs from V2C cells to those treated with EVs from 293T cells (*, P < .05; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Purification, Filtration, Concentration Assay, Incubation, Isolation, Glo Assay

Journal: The Journal of Infectious Diseases

Article Title: Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

doi: 10.1093/infdis/jiy472

Figure Lengend Snippet: Effect of cdk4/6 inhibitors on VP40-producing cell viability and extracellular vesicle biogenesis. 293T and V2CH cells were treated with low, medium, and high concentrations of Fascaplysin (0.1, 0.5, 1.0 µM) or Ribociclib (0.1, 1.0, 10.0 µM) for 5 days. DMSO was also used at a final concentration of 1%. (A) Supernatants were harvested, filtered (0.22 µm), and analyzed by ZetaView for concentration of particles. (B) Cell viability was also measured by CellTiter-Glo. Statistical analysis by Student’s 2-tailed t test compares drug-treated groups to untreated controls of their own cell type (*, P < .05; **, P < .01; ***, P < .001).

Article Snippet: Cdk4/6 inhibitors Fascaplysin (0.1–1 µM; Abcam) and Ribociclib (LEEO11; 0.1–10.0 µM; MedChemExpress) were used for the treatment of 293T and VP40 clone cell cultures for the analysis of cyclin D1 inhibition, cell viability, and EV production.

Techniques: Concentration Assay

Journal: Journal of Hepatocellular Carcinoma

Article Title: Exploration of the Mechanism of Action of Dendrobium officinale in the Treatment of Liver Cancer Based on Network Pharmacology, Molecular Docking and in vitro Validation

doi: 10.2147/JHC.S527095

Figure Lengend Snippet: The aqueous extract of Dendrobium officinale exerts an anti-liver cancer effect by down regulating the protein expression levels of p-PI3K/PI3K, AKT1, EGFR, and CCND1. The protein expression levels of ( A ) p-PI3K/PI3K, ( B ) AKT1, ( C ) EGFR, and ( D ) CCND1 were determined by Western blotting. DNL, DNM, DNH vs Control, * p <0.05, ** p <0.01, and *** p <0.001.

Article Snippet: CCK-8 (Elabsciencec, model: E-CK-A362); incubator and pipette gun were purchased from Thermo; AKT1 (model: 20584-1-AP), EGFR (model: 18986-1-AP), CCND1 (model: 26939-1-AP) antibodies were purchased from proteintech; PI3K (proteintech, model: 10176-2-AP); PI3K (proteintech, model: 10176-2-AP) antibodies were purchased from proteintech.

Techniques: Expressing, Western Blot, Control

Journal: Clinical and Translational Medicine

Article Title: ZC3H13‐mediated m6A stabilization of CCND1 promotes malignant progression and is associated with poor anti‐PD‐1 response in HNSCC

doi: 10.1002/ctm2.70750

Figure Lengend Snippet: Identification of CCND1 as a downstream transcript of ZC3H13 via MeRIP‐seq and transcriptomic analyses. (A) The most highly enriched m6A consensus motif identified from MeRIP‐seq data. (B) Density plot showing the distribution of identified m6A peaks across different regions of mRNA transcripts, including the 5′ untranslated region (5′ UTR), coding sequence (CDS), and 3′ untranslated region (3′ UTR). (C) Gene Ontology (GO) biological process enrichment analysis of genes with ZC3H13‐regulated m6A changes. (D and E) The mRNA and protein expression levels of cell cycle‐related genes (CDK1, CCNB1, CCNA2) following ZC3H13 knockdown (sh1, sh2) were assessed by RT‐qPCR (D) and Western blotting (E). (F and G) The expression changes of the aforementioned cell cycle‐related genes after ZC3H13 overexpression (OE) were determined by RT‐qPCR (F) and Western blotting (G). (G) Venn diagram showing the overlap between MeRIP‐seq‐identified candidate m6A‐regulated transcripts and genes enriched in the ZC3H13‐high post‐treatment malignant cell population, identifying 37 potential downstream candidates, including CCND1, CSTB, and NDUFB1. (I‐J) RT‐qPCR (I) and Western blot (J) validation of the suppressive effect of ZC3H13 knockdown on the expression of candidate target genes (CSTB, CCND1, NDUFB1). (K‐L) RT‐qPCR (K) and Western blot (L) validation demonstrating the promoting effect of ZC3H13 overexpression on target gene expression. (M) MeRIP‐qPCR assay quantifying the changes in m6A modification abundance on CCND1 mRNA following ZC3H13 depletion. (N) Integrative Genomics Viewer (IGV) browser tracks displaying the distribution and abundance of sequencing peaks along the CCND1 transcript in the control (ZC3H13) and knockdown (shZC3H13) groups. Data are presented as mean ± SD from three independent biological experiments unless otherwise indicated. * p < .05, ** p < .01, *** p < .00.

Article Snippet: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

Techniques: Sequencing, Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Over Expression, Biomarker Discovery, Targeted Gene Expression, Modification, Control

Journal: Clinical and Translational Medicine

Article Title: ZC3H13‐mediated m6A stabilization of CCND1 promotes malignant progression and is associated with poor anti‐PD‐1 response in HNSCC

doi: 10.1002/ctm2.70750

Figure Lengend Snippet: ZC3H13 enhances CCND1 mRNA stability in an IGF2BP1‐dependent manner. (A and B) Actinomycin D transcription inhibition assay followed by RT‐qPCR to assess the effect of ZC3H13 knockdown (sh) on the degradation rate and stability of CCND1 mRNA in HNSCC cells. (C) Polysome profiling analysis illustrating the distribution of monosomes and polysomes in control (Vector) and ZC3H13‐depleted cells. (D) Schematic illustration of the predicted m6A modification site on the CCND1 transcript and the construction of wild‐type and mutant dual‐luciferase reporter vectors. The mutant reporter was generated by an A‐to‐G substitution within the predicted m6A consensus motif, changing TGCCAG to TGCCGG. (E) Dual‐luciferase reporter assay evaluating the relative luciferase activity of WT or mutant CCND1 reporters following ZC3H13 overexpression (OE). (F) RNA pulldown assay followed by Western blotting to detect the direct binding of candidate m6A reader proteins (YTHDC1, YTHDF2, IGF2BP1, IGF2BP2) to different regions (5' UTR, CDS) of the CCND1 transcript. (G) RIP‐qPCR assay quantifying the specific enrichment of CCND1 mRNA by various m6A reader proteins. (H and I) Actinomycin D RNA decay assay showing the effect of IGF2BP1 knockdown on CCND1 mRNA stability. Accelerated CCND1 mRNA degradation after IGF2BP1 depletion supports IGF2BP1 as a reader protein that stabilizes CCND1 mRNA. Data are presented as mean ± SD from three independent biological experiments. Statistical tests were selected according to the experimental design as described in the section. * p < .05, ** p < .01, *** p < .001.

Article Snippet: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

Techniques: Inhibition, Quantitative RT-PCR, Knockdown, Control, Plasmid Preparation, Modification, Mutagenesis, Luciferase, Generated, Reporter Assay, Activity Assay, Over Expression, Western Blot, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: ZC3H13‐mediated m6A stabilization of CCND1 promotes malignant progression and is associated with poor anti‐PD‐1 response in HNSCC

doi: 10.1002/ctm2.70750

Figure Lengend Snippet: Clinical significance of CCND1 in HNSCC and its rescue effect on ZC3H13‐mediated cellular malignant phenotypes. (A) Representative immunohistochemistry (IHC) images demonstrating CCND1 expression in normal oral tissues, anti‐PD‐1 responder tissues, and anti‐PD‐1 non‐responder tumour tissues. (B) Statistical comparison of CCND1 IHC scores between normal and tumour tissues in a clinical cohort of 120 paired samples. (C) Differential analysis of CCND1 protein expression between anti‐PD‐1 responders (n = 38) and anti‐PD‐1 non‐responders ( n = 52) patients. (D) Comparison of CCND1 IHC scores stratified by histological grade (Grade 1–2 vs Grade 3–4). (E) Comparison of CCND1 IHC scores stratified by clinical stage (Stage 1–2 vs. Stage 3–4). (F) Kaplan–Meier survival curves showing the difference in overall survival (OS) between CCND1 high‐expression ( n = 55) and low‐expression ( n = 65) groups. Patients were dichotomized according to the median H‐score of the corresponding marker. (G) Pearson correlation analysis revealing a significant positive correlation between ZC3H13 and CCND1 protein expression (IHC scores) in HNSCC tumour tissues. (H) Western blot analysis of ZC3H13 and CCND1 protein expression levels in cells co‐transfected with Vector, sh_ZC3H13, oe_CCND1, or sh+oe to validate the efficacy of the rescue experiment. (I) CCK‐8 assay evaluating the rescue effect of CCND1 overexpression on the proliferation inhibition induced by ZC3H13 knockdown in HNSCC cells. (J and K) Colony formation assay and quantitative analysis evaluating the restorative effect of CCND1 on the long‐term proliferative capacity of ZC3H13‐depleted cells. (L and M) Transwell migration assay and quantitative analysis confirming that CCND1 reversed the suppressive effect of ZC3H13 knockdown on cell migration in vitro. (N and O) Transwell invasion assay and quantitative analysis verifying that CCND1 rescued the impaired invasive capability of cells induced by ZC3H13 knockdown. Data are presented as the mean ± SD from three independent experiments. * p < .05, ** p < .01, *** p < .001, ns indicates not significant.

Article Snippet: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

Techniques: Immunohistochemistry, Expressing, Comparison, Marker, Western Blot, Transfection, Plasmid Preparation, CCK-8 Assay, Over Expression, Inhibition, Knockdown, Colony Assay, Transwell Migration Assay, Migration, In Vitro, Transwell Invasion Assay

Journal: Clinical and Translational Medicine

Article Title: ZC3H13‐mediated m6A stabilization of CCND1 promotes malignant progression and is associated with poor anti‐PD‐1 response in HNSCC

doi: 10.1002/ctm2.70750

Figure Lengend Snippet: The ZC3H13/CCND1 axis remodels the HNSCC immune microenvironment and the proposed mechanistic model. (A) Gene Ontology (GO) enrichment analysis revealing biological pathways related to the negative regulation of cell activation and apoptosis. (B) Bar plot illustrating the changes in the proportion of CD4 + T cells between the Pre‐ and Post‐immunotherapy groups. (C) Boxplot comparing the quantitative CytoTRACE scores before and after immunotherapy (Pre vs. Post). (D) UMAP feature plots displaying the spatial distribution of cell differentiation states (CytoTRACE scores) in Pre‐ and Post‐treatment samples. (E) Violin plot demonstrating the significant difference in CD4+ T cell exhaustion scores between the Pre and Post treatment groups. (F) The relative mRNA expression levels of key immune checkpoint molecules (PDCD1, LAG3, CTLA4) in CCND1‐low and CCND1‐high groups were determined by RT‐qPCR. (G) Representative multiplex immunofluorescence images showing CCND1, CD4, PD‐1 and DAPI staining in mouse HNSCC tissues from control and ZC3H13‐deficient groups. Scale bar, 20 µm. (H) The quantification for the multiplex immunofluorescence (mIF) staining in the tumour microenvironment. (I) Schematic mechanistic model. Data are presented as the mean ± SD. *** p < .001.

Article Snippet: Sections were incubated with the indicated primary antibodies at room temperature for 1 h: CD4 (HY‐ P83756 , MCE, 1:500), CCND1 (HY‐ P80098 , MCE, 1:50), and PD‐1 (84651, Cell Signaling Technology, 1:100) in the optimized order of CD4, CCND1, and PD‐1.

Techniques: Activation Assay, Cell Differentiation, Expressing, Quantitative RT-PCR, Multiplex Assay, Immunofluorescence, Staining, Control

Journal: Journal of ethnopharmacology

Article Title: Dehydrocorydaline attenuates myocardial ischemia-reperfusion injury via the FoXO signalling pathway: A multimodal study based on network pharmacology, molecular docking, and experimental study.

doi: 10.1016/j.jep.2024.118738

Figure Lengend Snippet: Fig. 5. Prediction and validation of DHC and its potential targets by molecular docking and IHC analysis. (A) Molecular docking of STAT3 and DHC. (B) Molecular docking of MDM2 and DHC. (C) Molecular docking of CDK2 and DHC. (D) Molecular docking of PLK1 and DHC. (E) Molecular docking of CCND1 and DHC. (F) Binding of DHC to CCND1 as determined through microscale thermophoresis (MST). (G) Binding of DHC to MDM2 as determined through MST. (H) Binding of DHC to CDK2 as determined through MST. (I) Relative protein levels of p-FOXO1A (n = 3). (J) Representative IHC images of p-FOXO1A, CCND1, and p-MDM2. (K) Relative protein levels of CCND1 (n = 3). (L) Relative protein levels of p-MDM2 (n = 3). *indicates a significant difference compared with the sham group, # indicates a significant difference compared with the MIRI group: *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01. DHC: dehydrocorydaline, IHC: immunohistochemical, MIRI: myocardial ischemia-reperfusion injury.

Article Snippet: The microscale thermophoresis (MST) technique was performed to validate the interaction between CCND1 (#HY-P72119, MedChemExpress, Shanghai, China), CDK2 (#HY-P70014, MedChemExpress, Shanghai, China), MDM2 (#HY-P701593, MedChemExpress, Shanghai, China) and DHC(#HY-N0674, MedChemExpress, Shanghai, China).

Techniques: Biomarker Discovery, Binding Assay, Microscale Thermophoresis, Immunohistochemical staining

Journal: Journal of ethnopharmacology

Article Title: Dehydrocorydaline attenuates myocardial ischemia-reperfusion injury via the FoXO signalling pathway: A multimodal study based on network pharmacology, molecular docking, and experimental study.

doi: 10.1016/j.jep.2024.118738

Figure Lengend Snippet: Fig. 7. Effects of DHC on the expression of predicted targets in the in vitro model of H/R injury. (A) Representative fluorescence images of the TUNEL assay. Photographs were taken at × 400 magnification. (B) Percentage of TUNEL-positive cells in each group. (C) Western blot showing the protein expression of cleaved- caspase 3 and cleaved-caspase 8 in each group. (D) Western blot showing the protein expression of p-FOXO1A, FOXO1A, CCND1, p-MDM2, and MDM2 in each group. (E) Relative protein levels of cleaved-caspase 3 and cleaved-caspase 8 measured in western blots (n = 3). (F) Relative protein levels of p-FOXO1A, FOXO1A, and p- FOXO1A/FOXO1A measured in western blots (n = 3). (G) Relative protein levels of p-MDM2, MDM2, and p-MDM2/MDM2 measured in western blots (n = 3). (H) Relative protein levels of CCND1 measured in western blots (n = 3). * indicates a significant difference compared to the control group; # indicates a significant difference compared to the H/R model group: *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01. DHC: dehydrocorydaline, H/R: hypoxia/reoxygenation, TUNEL: TdT- mediated dUTP-biotin nick end labelling.

Article Snippet: The microscale thermophoresis (MST) technique was performed to validate the interaction between CCND1 (#HY-P72119, MedChemExpress, Shanghai, China), CDK2 (#HY-P70014, MedChemExpress, Shanghai, China), MDM2 (#HY-P701593, MedChemExpress, Shanghai, China) and DHC(#HY-N0674, MedChemExpress, Shanghai, China).

Techniques: Expressing, In Vitro, Fluorescence, TUNEL Assay, Western Blot, Control

Journal: Cell Death & Disease

Article Title: STAT3/miR-135b/NF-κB axis confers aggressiveness and unfavorable prognosis in non-small-cell lung cancer

doi: 10.1038/s41419-021-03773-x

Figure Lengend Snippet: A , B Activity of the NF-κB luciferase reporter gene in 293FT ( A ) transfected with miR-135b mimics, miR-135b inhibitors and corresponding negative control (NC) and in A549 stable cell lines ( B ). C , D Real-time PCR analysis of the mRNA expression of NF-κB downstream genes in the indicated cells. E Western blotting detecting the protein level of NF-κB downstream genes, Bcl-2, Bcl-xL, Cyclin D1, and MMP9 in indicated cells. β-tubulin served as a loading control. *Non-specific band. F Western blotting detecting the protein level of NF-κB signaling, p65, phospho-p65 (p-p65), IKKβ, phospho-IKKα/β (p-IKKα/β), in the indicated cells stimulated with TNF-α (10 ng/ml) for 20 min. β-tubulin served as a loading control. G Representative images of immunofluorescence staining of p65 (labeled with TRITC) in the indicated cells stimulated with TNF-α (10 ng/ml) for 20 min. Scale bars, 20 μm. Error bars represent the mean ± SD obtained from three independent experiments. p values are calculated by a two-tailed, unpaired t -test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies against Bcl-xl (1:1000, #2764, Cell Signaling Technology, USA), Bcl-2 (1:500, #2764, Cell Signaling Technology, USA), CCND1 (1:1000, TA801655, Origene, USA), CYLD (1:1000, #2978, Cell Signaling Technology, USA), β-tubulin (1:5000, RM2003, Ruikang, Beijing), NF-κB p65 (1:1000, #8242, Cell Signaling Technology, USA), Phospho-p65 (Ser536) (1:1000, #3033, Cell Signaling Technology, USA), IKKβ (1:1000, #2684, Cell Signaling Technology, USA), Phospho-IKKα/β (Ser176/180) (1:500, #2694, Cell Signaling Technology, USA), PARP (1:1000, #YM3132, Immunoway, USA), Cleaved Caspase-3(Asp175) (1:1000, #9661, Cell Signaling Technology, USA) were used.

Techniques: Activity Assay, Luciferase, Transfection, Negative Control, Stable Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Immunofluorescence, Staining, Labeling, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 target transcripts include mRNAs that encode proteins involved in myoblast proliferation and differentiation. ( A ) RIP assay using cytoplasmic lysates prepared from C2C12 cells using either anti-MYF5 antibody or IgG under conditions that preserved mRNA-RBP (mRNP) complexes. ( B ) Western blot analysis of MYF5 recovered in IP samples. ( C ) Following MYF5 RIP, MYF5-bound mRNAs were identified by microarray (RIP-chip) analysis in growing (GM) C2C12 cells. Data represent the Z -ratio of mRNAs in MYF5 RIP relative to IgG RIP. ( D ) RIP followed by RT-qPCR analysis to validate the association of MYF5 with mRNAs encoding myogenic proteins in proliferating C2C12 myoblasts; the levels of mRNAs in MYF5 IP were normalized to the levels of Gapdh mRNA and plotted as fold enrichment relative to the levels seen in control IgG IP samples. Discontinuous gray line: twofold enrichment in mRNAs bound to MYF5. ( E ) RIP analysis of Flag-MYF5 interaction with Ccnd1 mRNA. Forty-eight hours after C2C12 transfection with Flag-MYF5, RIP analysis was carried out using IgG or anti-Flag antibodies. Ccnd1 mRNA was detected by RT-qPCR analysis and its levels in Flag IP were compared with those in control IgG IP samples; Actn mRNA (encoding the housekeeping protein β-Actin) was measured to normalize sample input. Data in (D,E) represent the means and S.E.M. from three or more independent experiments. ( F ) Ingenuity pathway analysis (IPA) of mRNAs enriched in MYF5 IP relative to IgG in C2C12 growing myoblasts. *, P < 0.05 and **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Western Blot, Microarray, RNA Immunoprecipitation - Chromatin Immunoprecipitation, Quantitative RT-PCR, Control, Transfection

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 binds to specific sequences on the Ccnd1 mRNA. (A) Top , schematic showing biotinylated RNA fragments spanning the 5′UTR, coding region (CR), and 3′UTR of Ccnd1 mRNA used for pulldown. Bottom , biotinylated RNA fragments were incubated with cytoplasmic lysates from C2C12 cells (GM); after pulldown using streptavidin beads, the levels of MYF5 bound to the biotinylated RNA segments were detected by western blot analysis. (B) Recombinant purified His-MYF5 was incubated with biotinylated Ccnd1 RNA fragments followed by pulldown and detection of MYF5 by western blot analysis using anti-MYF5 antibody. (C) Schematic of biotinylated RNA fragments spanning the Ccnd1 3′UTR-C transcript ( top ), were tested for binding to His-MYF5 after pull-down using streptavidin beads; His-MYF5 interaction with RNA segments of fragment C ( middle ) and smaller RNAs after closer subdivision of fragments 9 through 11 ( bottom ) were assessed by western blot analysis using anti-MYF5 antibody. (D) GST or GST-MYF5 were incubated with radiolabeled Ccnd1 3′UTR-C10–1, then either resolved on native acrylamide gels ( left ) by RNA electrophoretic mobility shift assay (EMSA), or crosslinked by UV irradiation and resolved by SDS-PAGE ( right ). (E) The domain of MYF5 that interacts with the Ccnd1 3′-C fragment was mapped by creating GST-tagged truncations of MYF5 ( left ) and testing their interaction by biotin pulldown and western blot analysis using anti-GST antibody ( right ).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Incubation, Western Blot, Recombinant, Purification, Binding Assay, Electrophoretic Mobility Shift Assay, Irradiation, SDS Page

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 regulates CCND1 expression in C2C12 myoblasts. (A) Western blot analysis of MYF5 and CCND1 expression during C2C12 differentiation; heat shock protein 90 (HSP90) was included as loading control. (B,C) Forty-eight hours after transfecting C2C12 cells with MYF5 siRNA or Ctrl siRNA, the levels of MYF5, CCND1 and loading control GAPDH were analyzed by western blot analysis (B) and the levels of Ccnd1 pre-mRNA and mRNA by RT-qPCR analysis (C). (D–F) Forty-eight hours after transfection of proliferating C2C12 myoblasts with Ctrl or CCND1 siRNAs, the levels of CCND1 and HSP90 were assessed by western blot analysis (D) and cell numbers were measured using a TC10 automated cell counter (BioRad) and represented as fold change in cell number after CCND1 silencing relative to those in the Ctrl siRNA group (E). At day 6 in differentiation medium (DM6) C2C12 differentiation was monitored by measuring creatine kinase activity (F). (G) Forty-eight hours after MYF5 was overexpressed using pFlag-MYF5 pcDNA3, the levels of MYF5, CCND1 and loading control HSP90 were studied by western blot analysis. Data in (B-F) represent the means and S.E.M. from three or four independent experiments. *, P < 0.05 and **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Transfection, Activity Assay

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: MYF5 promotes translation of Ccnd1 mRNA. (A,B) Forty-eight hours after Ctrl or MYF5 siRNA transfection of C2C12 cells, cytoplasmic extracts were fractionated through sucrose gradients to obtain cytoplasmic components of progressively larger weight: ribosomal subunits (40S, 60S), monosomes (80S) and low-molecular-weight (LMW) and high-molecular-weight (HMW) polysomes (A). The relative distribution of Gapdh mRNA, encoding a housekeeping protein, and Ccnd1 mRNA were measured by RT-qPCR analysis of RNA in each of the gradient fractions and represented as percentage of total RNA in the gradient (B). (C) Schematic of the dual luciferase reporter plasmids derived from the parent vector psiCHECK2 (psi), which expresses renilla luciferase (RL) and the internal control firefly luciferase (FL), and psiCHECK2-derived plasmids bearing the Ccnd1 fragments downstream of the RL coding region. (D) Top , Influence of MYF5 silencing on the expression of the reporter constructs. Twenty-four h after transfection of C2C12 cells with either MYF5 siRNA or Ctrl siRNA, each reporter plasmid was transfected, and 16 h later the ratio of RL activity to FL activity was measured. The decrease in relative RL/FL ratio of MYF5 siRNA-transfected cells relative to the RL/FL ratio of Ctrl siRNA-transfected cells is indicated. Bottom , RT-qPCR analysis of RL mRNA levels normalized to FL mRNA levels in each transfection group. Data represent the means and S.E.M. from 3 independent experiments. N.S., not significant; *, P < 0.05; **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Transfection, Molecular Weight, High Molecular Weight, Quantitative RT-PCR, Luciferase, Derivative Assay, Plasmid Preparation, Control, Expressing, Construct, Activity Assay

Journal: Nucleic Acids Research

Article Title: Novel RNA-binding activity of MYF5 enhances Ccnd1 / Cyclin D1 mRNA translation during myogenesis

doi: 10.1093/nar/gkw023

Figure Lengend Snippet: Influence of MYF5 on myogenesis via regulation of CCND1 expression. (A,B) C2C12 cells were transfected with MYF5 siRNA or Ctrl siRNA, along with a control vector [pcDNA3-Flag (pV)] or a plasmid vector that expressed Myc-tagged CCND1. Forty-eight hours later, the levels of CCND1 and MYF5 were analyzed by western blot analysis (A) and the degree of differentiation was analyzed by measuring creatine kinase activity at day 6 into differentiation (B). Data presented are the means and S.E.M. from four independent experiments; significance (P) is indicated. (C) Proposed model whereby MYF5 modulates myogenesis by acting upon CCND1 expression on two levels: first, MYF5 activates Ccnd1 transcription moderately, and second, MYF5 binds the Ccnd1 mRNA at the CR and 3′UTR, promoting Ccnd1 mRNA translation. The net effect is a CCND1-mediated increase in myoblast proliferation necessary at the initiation of myogenesis. N.S., not significant; *, P < 0.05; **, P < 0.01 (Student's t -test).

Article Snippet: CCND1 siRNA (SR408533, Origene) was transfected 48 h before measuring cell number.

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Activity Assay