Journal: Drug Design, Development and Therapy

Article Title:

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells

doi: 10.2147/dddt.s264122

Figure Lengend Snippet: Figure 3 Acetylsalicylic acid (ASA) inhibits sialidase activity in live epidermal growth factor (EGF)-stimulated pancreatic cancer PANC-1 and MiaPaCa-2 cells using fluorescent microscopy and quantitative spectrophotometry analyses. Live cell sialidase assays were performed to detect the effect of aspirin pre-treatment on sialidase activity. Live cells were pre-treated with increasing concentrations of ASA (1.6 mM– 6.4 mM), followed with 30 ng/mL of EGF to initiate sialidase activity. 0.318 mM of 2ʹ- (4-methylumbbelliferyl) α-d-N-acetylneuraminic acid (4-MUNANA) substrate was added to quantify neuraminidase-1 activity. (A) PANC-1 live cell sialidase activity. Blue fluorescence is a direct measure of Neu-1 activity. (B) Densitometry analysis of fluorescent images in A using ImageJ software and presented as a bar graph. 50 points were taken surrounding cells for each image. Graphs represent 50-point analysis of two or more images. (C) Quantitative spectrophotometry analysis of the effect of ASA pre- treatment on sialidase activity in EGF-stimulated live PANC-1 pancreatic cancer cells. (D) MiaPaCa-2 fluorescent and phase images were taken within 2 minutes using an epi- fluorescent microscope (200×). Scale bar represents 50 μm. (E) Quantitative spectrophotometry analysis of the effect of aspirin pre-treatment on sialidase activity in EGF- stimulated live MiaPaCa-2 pancreatic cancer cells. Significance is represented in comparison to the EGF-stimulated cells as a control. Error bars represent ± SEM from three separate experiments performed in triplicates (n=3). Significance is represented in comparison to the EGF-stimulated cells as a control by one-way ANOVA using the uncorrected Fisher’s LSD multiple comparisons test with 95% confidence with indicated asterisks for statistical significance, ***p = 0.0007, ****p ≤ 0.0001, n = 3. Abbreviations: ASA, acetylsalicylic acid; EGF, epidermal growth factor; 4-MUNANA, 2ʹ-(4-methylumbbelliferyl) α-d-N-acetylneuraminic acid; SEM, standard error of the mean.

Article Snippet: Cells were stimulated with 100 ng/mL EGF (CL-100-26, Cedarlane) for 30 minutes, or left unstimulated as a control.

Techniques: Activity Assay, Microscopy, Spectrophotometry, Fluorescence, Software, Comparison, Control

Journal: Drug Design, Development and Therapy

Article Title:

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells

doi: 10.2147/dddt.s264122

Figure Lengend Snippet: Figure 4 Celecoxib inhibits sialidase activity in live epidermal growth factor (EGF)-stimulated pancreatic PANC-1 cancer cells using fluorescent microscopy but not with quantitative spectrophotometry analyses. Live cell sialidase assays were performed to detect the effect of celecoxib pre-treatment on sialidase activity. Live PANC-1 cells were pre-treated with increasing concentrations of celecoxib (0.1–0.8 mM) followed by stimulation with 30 ng/mL of EGF to initiate sialidase activity. 0.318 mM of 2ʹ- (4-methylumbbelliferyl) α-d-N-acetylneuraminic acid (4-MUNANA) substrate was added to quantify Neu-1 activity. (A) Fluorescent and phase images were taken within 2 minutes of celecoxib exposure using an epi-fluorescent microscope (200×). Scale bar represents 50 μm. Blue fluorescence is a direct measure of Neu-1 activity. (B) Densitometry analysis of fluorescent images using ImageJ software and presented as a bar graph. 50 points were taken surrounding cells for each image. Graphs represent a 50-point analysis of two or more images. Significance is represented in comparison to the EGF-stimulated cells as a control (C) Quantitative spectrophotometry analysis of the effect of celecoxib pre-treatment on sialidase activity in EGF-stimulated live PANC-1 pancreatic cancer cells. Error bars represent ± SEM from three separate experiments performed in triplicates (n=3). Significance is represented in comparison to the EGF-stimulated cells as a control by one-way ANOVA using the uncorrected Fisher’s LSD multiple comparisons test with 95% confidence with indicated asterisks for statistical significance, ****p ≤ 0.0001, n = 3. Abbreviations: EGF, epidermal growth factor; 4-MUNANA, 2ʹ-(4-methylumbbelliferyl) α-d-N-acetylneuraminic acid; SEM, standard error of the mean.

Article Snippet: Cells were stimulated with 100 ng/mL EGF (CL-100-26, Cedarlane) for 30 minutes, or left unstimulated as a control.

Techniques: Activity Assay, Microscopy, Spectrophotometry, Fluorescence, Software, Comparison, Control

Journal: Drug Design, Development and Therapy

Article Title:

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells

doi: 10.2147/dddt.s264122

Figure Lengend Snippet: Figure 5 Acetylsalicylic acid (ASA) and celecoxib inhibit Clostridium perfringens neuraminidase activity. Sialidase assays were performed using isolated neuraminidase (Clostridium perfringens; with a specific activity of 1 unit (U) per 1.0 mmole of N-acetylneuraminic acid per minute). Reconstituted neuraminidase in reaction buffer was exposed to 0.318 mM of its fluorogenic substrate 2ʹ-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA) alone or in combination with increasing concentra tions of (A) ASA or (B) celecoxib. Enzyme activity was analyzed with a spectrophotometer to quantify relative fluorescence at 450 nm. Relative fluorescence is presented as a bar graph. Error bars represent ± SEM from three separate experiments performed in triplicates (n=3). Significance is represented in comparison to the EGF-stimulated cells as a control by one-way ANOVA using the uncorrected Fisher’s LSD multiple comparisons test with 95% confidence with indicated asterisks for statistical significance, *p ≤ 0.01, ****p ≤ 0.0001, n = 3. Abbreviations: ASA, acetylsalicylic acid; 4-MUNANA, 2ʹ-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid; SEM, standard error of the mean.

Article Snippet: Cells were stimulated with 100 ng/mL EGF (CL-100-26, Cedarlane) for 30 minutes, or left unstimulated as a control.

Techniques: Activity Assay, Isolation, Spectrophotometry, Fluorescence, Comparison, Control

Journal: Drug Design, Development and Therapy

Article Title:

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells

doi: 10.2147/dddt.s264122

Figure Lengend Snippet: Figure 6 Acetylsalicylic acid (ASA) inhibits Neu-1 cleavage of α-2,3 sialic acid in PANC-1 cells. PANC-1 cells were pre-treated with anti-Neu-1 antibody, 3.2 mM, 4.8 mM, or 6.4 mM of ASA for 1 hour, or left untreated as a control. Cells were then stimulated with 1 ug/mL EGF for 5, 10, 15, or 30 minutes, or left untreated as a control. Cells were fixed, permeabilized and immunostained for α-2,3 sialic acid using biotinylated MAL II. Cells were stained with DAPI-containing mounting media to visualize the nuclei. (A) Stained cells were visualized by epi-fluorescent microscopy using a 200x objective. Scale bar represents 50 μm. (B) Quantitative analysis of MAL II expression levels by relative density corrected for average background staining of the Streptavidin 594 secondary antibody. Data shown for conditions stimulated with EGF for 30 minutes. Each bar in the figure represents the mean corrected for density staining ± SEM due to multiple images being quantified (3–4). The data are a combination of two independent experiments with similar results. (C) Flow cytometry was used to confirm the expression levels of α-2,3 sialic acid. Graphs represent an overlay of treated cells, untreated cells, stimulated cells, secondary control, and autofluorescence control. (D) Graph comparing expression levels of α-2,3 sialic acid under indicated conditions from flow cytometry analysis. Abbreviations: ASA, acetylsalicylic acid; EGF, Epidermal growth factor; MAL II, Maackia amurensis lectin II; SEM, standard error of the mean.

Article Snippet: Cells were stimulated with 100 ng/mL EGF (CL-100-26, Cedarlane) for 30 minutes, or left unstimulated as a control.

Techniques: Control, Staining, Microscopy, Expressing, Flow Cytometry

Journal: Drug Design, Development and Therapy

Article Title:

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells

doi: 10.2147/dddt.s264122

Figure Lengend Snippet: Figure 7 Acetylsalicylic acid (ASA) inhibits phosphorylation of the EGFR in PANC-1 and MiaPaCa-2 cells. PANC-1 and MiaPaCa-2 cells were pretreated with 3.2 mM, 4.8 mM, or 6.4 mM of aspirin for 1 hour, or left untreated as a control. Cells were then stimulated with 100 ng/mL EGF for 30 minutes or left untreated as a control. Cells were fixed, permeabilized, and immunostained for EGFR and phosphorylated EGFR (pEGFR). DAPI-containing mounting media was used to visualize the nuclei. (A) Stained PANC- 1 cells, (B) quantitative analysis of pEGFR (Tyr1173) and EGFR expression in PANC1 cells by relative density corrected for average background staining antibody and (C) the ratio of pEGFR to EGFR expression for PANC-1. (D) Stained MiaPaCa-2 cells were visualized by epi-fluorescent microscopy using a 100x objective or 200x objective. (E) Quantitative analysis of pEGFR (Tyr1173) and EGFR by relative density corrected for average background staining antibody in MiaPaCa-2 cells. (F) The ratio of pEGFR to EGFR expression for MiaPaCa-2 cells is shown. Each bar in the figure represents the mean corrected for density staining ± SEM due to multiple images being quantified (3–4). Abbreviations: ASA, acetylsalicylic acid; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; pEGFR, phosphorylated epidermal growth factor receptor; DAPI, 4′,6-diamidino-2-phenylindole; SEM, standard error of the mean.

Article Snippet: Cells were stimulated with 100 ng/mL EGF (CL-100-26, Cedarlane) for 30 minutes, or left unstimulated as a control.

Techniques: Phospho-proteomics, Control, Staining, Expressing, Microscopy

Journal: MedComm

Article Title: Identification of circulating T‐cell immunoglobulin and mucin domain 4 as a potential biomarker for coronary heart disease

doi: 10.1002/mco2.320

Figure Lengend Snippet: Serum sTIMD4 levels were increased associated with CHD. Serum samples collected from healthy controls, CHD, CCS, or ACS patients were used to determine TIMD4 levels by ELISA, followed by comparison with nonparametric tests between healthy controls and CHD patients (Group 1, A) or CCS and ACS patients (Group 2, B). Each dot presents an individual. The whisker plot presents the mean with min to max, and the beard plot presents an average from smallest to largest. (C) ROC curve of serum sTIMD4 for predicting CHD events. AUC is 0.787. The optimal cut‐off is 0.34. The sensitivity is 0.749, and the specificity is 0.667.

Article Snippet: The level of serum sTIMD4 levels were measured using a human TIMD4 ELISA kit (Cat# CSB‐EL023546HU; Cusabio, Wuhan, China) following the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Whisker Assay

Journal: MedComm

Article Title: Identification of circulating T‐cell immunoglobulin and mucin domain 4 as a potential biomarker for coronary heart disease

doi: 10.1002/mco2.320

Figure Lengend Snippet: Ox‐LDL increased ADAM17 and sTIMD4 while decreased mTIMD4 level in macrophages. RAW264.7 cells were treated with ox‐LDL at the indicated concentrations for 24 h (A, D–G) or with 80 μg/mL ox‐LDL for the indicated times (B) or LPS at 1 μg/mL for 24 h (C). After treatment, total cellular proteins, RNA, and treatment mediums were collected. (A–C) Expression of ADAM17, mTIMD4, IL‐6, IL‐10, pro‐IL‐1β, IL‐1β, caspase‐1, and IL‐18 in cells and sTIMD4 in medium was detected by Western blotting ( n = 3). (D and E) Expression of TIMD4, ADAM17, TNF‐α, and IL‐6 mRNA was determined by qRT‐PCR ( n = 3–4). HSP90 was used as a loading control. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: The level of serum sTIMD4 levels were measured using a human TIMD4 ELISA kit (Cat# CSB‐EL023546HU; Cusabio, Wuhan, China) following the manufacturer's instructions.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: MedComm

Article Title: Identification of circulating T‐cell immunoglobulin and mucin domain 4 as a potential biomarker for coronary heart disease

doi: 10.1002/mco2.320

Figure Lengend Snippet: Ox‐LDL/LPS activated ADAM17 to increase cleavage of mTIMD4 in macrophages. RAW264.7 cells treated with ox‐LDL (80 μg/mL) and TAPI‐1 (1 μM) for 24 h. (A) and (B) mTIMD4 expression was detected by immunofluorescent staining ( n = 5). (C–F) The protein expression of mTIMD4, sTIMD4 in medium, TLR‐4, p‐NF‐κB, NF‐κB, IL‐6, caspase‐1, pro‐IL‐1β, TGF‐β1, and IL‐10 were detected by Western blotting ( n = 3). (G–J) The mRNA expression mTIMD4, TLR‐4, IL‐6, and TGF‐β1 was determined by qRT‐PCR ( n = 3–4). (K–N) RAW264.7 cells were treated with LPS (1 μg/mL) and TAPI‐1 (1 μM) for 24 h, and the protein expression of mTIMD4, sTIMD4 in medium, TLR‐4, p‐NF‐κB, NF‐κB, IL‐6, caspase‐1, pro‐IL‐1β, TGF‐β1, and IL‐10 were detected by Western blotting ( n = 3). (O–P) RAW264.7 cells were transfected with 20 nM control si‐NC or si‐TIMD4 for 48 h and incubated with ox‐LDL (80 μg/mL) and TAPI‐1 (1 μM) for 24 h, followed by determination of mTIMD4, TLR‐4, p‐NF‐κB, NF‐κB, IL‐6, IL‐10, TGF‐β1, caspase‐1, pro‐IL‐1β protein expression by Western blotting ( n = 3). HSP90 or GAPDH was used as a loading control. ns, not significant. * p < 0.05; ** p < 0.01.

Article Snippet: The level of serum sTIMD4 levels were measured using a human TIMD4 ELISA kit (Cat# CSB‐EL023546HU; Cusabio, Wuhan, China) following the manufacturer's instructions.

Techniques: Expressing, Staining, Western Blot, Quantitative RT-PCR, Transfection, Control, Incubation

Journal: bioRxiv

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1101/2022.10.29.514376

Figure Lengend Snippet: a , VEGFA site 2 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in WTC-11 iPSCs. b-c , Genome browser visualization of MRE11 enrichment at an (b) on-target and (c) representative off-target position with 4 mismatches (‘4 mm’) in WTC-11 iPSCs with Cas9 targeting VEGFA site 2 . DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). d , Schematic of the DISCOVER-Seq+ protocol in the knock-in of a cancer neoantigen-specific, transgenic TCR (tgTCR) into the TRA locus of primary human T cells. e , TRA Cas9 target sites in primary T cells detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right). f-g , Genome browser visualization of MRE11 enrichment at two representative 4-mismatch (‘4mm’) off-target positions in primary human T cells with Cas9 targeting TRA for knock-in of a tgTCR template. DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). h-i , (h) Plots of MRE11 ChIP-seq reads-per-million enrichment within a 1.5 kb window for samples with (y-axis) or without (x-axis) DNA-PKcs inhibition, at all TRA Cas9 off-target sites in primary human T cells from the DNA-PKcs inhibited samples. Each point in the plot (20 total) corresponds to a putative target site. Significant differences (p < 1E-3 or p < 1E-5) between y-axis and x-axis values were determined using two-sided Wilcoxon rank sum test. (i) Same as panel h, for cells delivered with Cas9 but without gRNA (negative control). j , TRA Cas12a (Cpf1) target sites in primary T cells. The results were the same between DISCOVER-Seq and DISCOVER-Seq+; only the on-target site was detected.

Article Snippet: Purified CD3+ T cells were activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher) at a 1:2 bead-to-cell ratio in RPMI-1640 (ATCC) supplemented with 10% fetal bovine serum (HyClone Defined), 100 units/mL Penicillin (Gibco), 100 ug/mL Streptomycin (Gibco), 100 IU/mL recombinant human IL-2 (Proleukin, Prometheus Laboratories) and 5 ng/mL recombinant human IL-7 (BioLegend) at 37C, 5% CO 2 .

Techniques: Knock-In, Transgenic Assay, ChIP-sequencing, Inhibition, Negative Control