Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Ascites in peritoneal carcinomatosis (PC) is biologically active (A) Kaplan-Meier curve showing survival of PC patients who underwent cytoreductive surgery (n = 121), stratified by the presence or absence of ascites. (B and C) Proliferation curves of (B) Colo-205 and (C) SNU-C1 cells treated with varying concentrations of ascites, measured by CellTitre-Glo assay. Data are represented as cell viability at day 5 relative to day 0. Dotted line represents cell viability of Colo-205 treated with 10% fetal bovine serum (FBS) at day 5 relative to day 0. Graph shows mean ± SD. (D and E) Migration of (D) Colo-205 and (E) SNU-C1 cells pre-treated with serum-free media (SFM), 10% FBS medium or 5% cell-free ascites (CFA) medium, assessed by transwell migration assay. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with SFM and 10% FBS or 5% CFA was denoted by ∗. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with 10% FBS and SFM or 5% CFA was denoted by # . # p < 0.05, ## p < 0.01, ### p < 0.001. Graph shows mean ± SD. (F) Enriched signaling pathways in colorectal PC cell lines upon treatment with CFA, identified by comparing gene expressions of cells treated with 5% versus 0.1% CFA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G and H) Treatment of (G) Colo-205 and (H) SNU-C1 cells with 5% CFA activated STAT3 signaling pathway through phosphorylation at Tyr705.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Glo Assay, Migration, Transwell Migration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Identification and validation of prognostic putative activators of STAT3 signaling in ascites (A) Workflow to identify clinically significant putative secreted STAT3 activators. (B) Kaplan-Meier survival curve illustrating poorer survival in patients expressing three biomarkers as compared to patients expressing 0–2 biomarkers. (C) Characterization of cytokine profiles in colorectal PC ascites and benign ascites. Top 25% most highly abundant cytokines in colorectal PC ascites (by mean abundance) are shown. Heatmap displays Z score of normalized mean pixel density of duplicate cytokine spots on the array. (D) Downregulated signaling pathways upon PAI-1 inhibition in Colo-205 cells exposed to CFA with high PAI-1 levels (PC085), CFA with low PAI-1 levels (PC249) and no PAI-1 (FBS control), identified using RNA microarray. Only signaling pathways with differential downregulation are presented. IL6-JAK-STAT3 signaling pathway was significantly downregulated in high PAI-1 CFA-treated cells upon PAI-1 inhibition. Normalized enrichment scores <0 indicate pathway suppression and scores >0 indicate pathway activation. (D) is representative of two independent biological experiments. ∗p < 0.05.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Expressing, Inhibition, Microarray, Activation Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Correlating PAI-1 level in ascites and intracellular STAT3 activation in cancer cells revealed distinct subgroups associated with differing susceptibility to PAI-1 inhibition (A) PAI-1 prevalence in colorectal PC ascites (n = 54). (B) Correlation between colorectal PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.4691, p = 0.0003). (C) PAI-1 prevalence in ascites of various histological PC subtypes (n = 156). † indicates benign ascites. (D) Correlation between various histological PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.6476, p < 0.0001). (A–D) PAI-1 concentrations are plotted on a log 2 scale to transform skewed data to normal distribution. p-STAT3(Y705) level was shown as optical density reading at 450 nm (OD450). (E) Untransformed values of PAI-1 and p-STAT3(Y705) levels from (D) were used for stratification strategy to identify patient subpopulations who might benefit from PAI-1 inhibition. Using 20 ng/mL PAI-1 level and 0.2 OD450 p-STAT3(Y705) level as cut-off values, three distinct subgroups of samples were observed: (i) high PAI-1 and high p-STAT3 levels, termed PAI-1 paracrine addicted (PPA) group (yellow region), (ii) low PAI-1 and high p-STAT3 levels, termed co-activators predominant (CAP) group (pink region) and (iii) low PAI-1 and low p-STAT3 levels, termed alternative pathways activation (APA) group (blue region). Each dot represents one patient ascites. Colors in each panel represent the various histological PC subtypes. All histological subtypes with less than five samples are grouped into other histological subtypes.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Activation Assay, Inhibition

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Cells dependent on PAI-1 to activate STAT3 are most susceptible to PAI-1 inhibition (A) Effect of TM5441 (PAI-1 inhibitor) on CFA-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow), CAP group (pink), APA group (blue) and FBS (control, black) demonstrated a left shift, indicating responsiveness to PAI-1 inhibition. (B) Differential sensitivity to TM5441 corresponding to the three subgroups, with PPA (n = 18) being the most sensitive to PAI-1 inhibition, followed by CAP (n = 59) and APA (n = 17). Graph shows mean ± SD. (C–E) Effect of (C) Napabucasin (STAT3 inhibitor), (D) BEZ235 (dual PI3K/mTOR inhibitor) and (E) Mitomycin C (conventional chemotherapeutic agent – DNA crosslinker) on the three subgroups of ascites-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow; n = 3), CAP group (pink; n = 3), APA group (blue; n = 1) and FBS (control, black). Targeting PAI-1, a dominant paracrine factor in ascites, was more effective than targeting downstream signaling pathway activated by ascites, proliferation pathway or DNA synthesis. (F) Evaluation of STAT3 suppression in Colo-205 cells treated with PPA CFA (PC085 and PC383), CAP CFA (PC249), APA CFA (PC010) and various concentrations of TM5441 by ELISA. STAT3 activation was shown as p-STAT3(Y705) and total STAT3 ratio at the indicated concentration relative to DMSO vehicle. Cells exposed to PPA CFA relied on PAI-1 to activate STAT3 as they required lower concentrations of TM5441 to suppress STAT3 activation. Graph shows mean ± SEM. Data in (A-E) are representative of at least three independent biological experiments and (F) is representative of two independent biological experiments. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Inhibition, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet:

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Sequencing, Microarray, Cell Culture, Software

Journal: Virology

Article Title: Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: insights on viral regulation of apoptosis and proliferation.

doi: 10.1016/j.virol.2007.09.016

Figure Lengend Snippet: Fig. 1. Time course study of S2-induced apoptosis in Vero E6 cells. (a) Morphology of Vero E6 cells under light microscope (40×) after rAd-Ctrl or rAd-S2 transductions from 0 to 96 h p.t. in 12- to 24-h intervals. Representative figures of three independent experiments are shown. (b) Cell viability along the time course is estimated quantitatively with Trypan blue exclusion assay. (c) The percentage of rAd-Ctrl or rAd-S2-transduced cells showing chromatin condensation was counted under fluorescence microscope after Hoechst 33342 staining. For both panels b and c, average of three independent experiments is shown with standard error of the mean (SEM). (d) Agarose gel electrophoresis showing the characteristic DNA laddering pattern resulting from internucleosomal DNA cleavage in rAd-S2-transduced cells in four selected time points. Three micrograms of low molecular weight DNA was loaded into each well. The ladders with 200 bp increments are indicated with arrowheads. The result is the representative of three independent experiments. ⁎pb0.01 when compared with cells transduced by rAd-Ctrl under the same condition.

Article Snippet: HEK293-derived AD-293 cells (Stratagene) used to propagate recombinant adenoviruses (rAds), and the African green monkey kidney cell line Vero E6 cells (CRL-1586, American Type Culture Collection) used for adenoviral transduction and microarray analysis were cultured and maintained as described (Chow et al., 2005).

Techniques: Light Microscopy, Trypan Blue Exclusion Assay, Fluorescence, Microscopy, Staining, Agarose Gel Electrophoresis, DNA Laddering, Molecular Weight

Journal: Virology

Article Title: Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: insights on viral regulation of apoptosis and proliferation.

doi: 10.1016/j.virol.2007.09.016

Figure Lengend Snippet: Fig. 3. Bcl-xL blocks S2-induced apoptosis in Vero E6 cells. Different dosage of rAd-Bcl-xL was co-infected with 50 MOI of rAd-S, -S1, -S2 or rAd-Ctrl and the effect on cell death and apoptosis induced by the rAds were assayed through (a) Trypan blue exclusion assay and (b) Hoechst 33342 staining at day 5 and day 3 p.t. respectively. Both figures indicated the average of three independent experiments with SEM.

Article Snippet: HEK293-derived AD-293 cells (Stratagene) used to propagate recombinant adenoviruses (rAds), and the African green monkey kidney cell line Vero E6 cells (CRL-1586, American Type Culture Collection) used for adenoviral transduction and microarray analysis were cultured and maintained as described (Chow et al., 2005).

Techniques: Infection, Trypan Blue Exclusion Assay, Staining

Journal: Virology

Article Title: Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: insights on viral regulation of apoptosis and proliferation.

doi: 10.1016/j.virol.2007.09.016

Figure Lengend Snippet: Fig. 4. S2-mediated inhibition of cell proliferation in Vero E6 cells. Vero E6 cells were transduced with the indicated rAds and their proliferations were estimated with MTTcell proliferation assay and are expressed as percentage of proliferation of mock-infected cells. Time- and dose-dependent anti-proliferation effect of S2 was shown in panels a and b, respectively. The time-dependent effect was performed with rAd of 25 MOI and cells in the dose-dependent experiment were collected at 3 days p.t. Results shown are the average of three independent experiments with SEM. ⁎pb0.01 when compared with cells transduced by rAd- Ctrl under the same condition.

Article Snippet: HEK293-derived AD-293 cells (Stratagene) used to propagate recombinant adenoviruses (rAds), and the African green monkey kidney cell line Vero E6 cells (CRL-1586, American Type Culture Collection) used for adenoviral transduction and microarray analysis were cultured and maintained as described (Chow et al., 2005).

Techniques: Inhibition, Transduction, Proliferation Assay, Infection

Journal: Cell metabolism

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

doi: 10.1016/j.cmet.2018.10.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 50 μg of total protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes, which were then probed with the following antibodies: anti-TFAM IgG HPA040648, anti-mtSSB IgG HPA002866, anti-ATP5B IgG HPA001520 (all Sigma), anti-POLG IgG PA5-35163, anti-cyclin E IgG MA5-14336, anti-mtCO4 IgG PA5-29992 (all Thermo Fisher Scientific), anti-UMPS IgG sc-398086 (Santa Cruz Biotechnology), anti-CAD IgG 93925, anti-p(S1859)-CAD IgG 12662 (both Cell Signaling), anti-NDUFS3 IgG ab110246, anti-NDUFA9 IgG ab14713, anti-mtCO1 IgG Ab14705, anti-SDHB IgG ab14714 (all Abcam); anti-DHODH IgG was either from Santa Cruz Biotechnology (sc-166348) or from Proteintech (14877-1-AP).

Techniques: Glo Assay, Pyruvate Assay, Bicinchoninic Acid Protein Assay, Labeling, Recombinant, Microarray, Clone Assay, Binding Assay, Software, Imaging, Protease Inhibitor, DNA Extraction, SYBR Green Assay

Journal: bioRxiv

Article Title: The mitochondrial-targeted peptide therapeutic elamipretide improves cardiac and skeletal muscle function during aging without detectable changes in tissue epigenetic or transcriptomic age

doi: 10.1101/2024.10.30.620676

Figure Lengend Snippet: A. PCA of DNA methylation (DNAm) microarray samples. PCA was performed following data normalization in SeSAMe and filtering of failed probes. B. Mean DNAm levels. Mean DNAm was estimated by taking the means of the filtered beta values for each sample. P-values were determined by two-way ANOVA and Tukey’s post-hoc test. P-values for the most relevant comparisons are shown in text. Lines represent sample means. C. Effect of ELAM treatment and aging on epigenetic age (DNAmAge) of mouse hearts. P-values were determined by two-way ANOVA and Tukey’s post-hoc test. P-values for the most relevant comparisons are shown in text. Error bars represent sample means ± standard deviations. D. Effect of ELAM treatment and aging on protein expression of cap-independent translation (CIT) targets. Upper panels: Western blot images of CIT targets Hsp70, TFAM, and MGMT alongside a loading control (β-actin). Lower panels: Quantification of the protein levels of CIT targets. P-values were determined by two-way ANOVA and Tukey’s post-hoc test. P-values for the most relevant comparisons are shown in text. Error bars represent sample means ± standard deviations.

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 10% dry milk in TBS-T, and incubated at 4°C overnight with the following antibodies (all at a dilution factor of 1:1000): mouse anti-β-actin (Santa-Cruz Biotechnology, Dallas, TX) and rabbit anti-TFAM, anti-MGMT, and anti-Hsp70 (Cell Signaling Technology, Danvers, MA).

Techniques: DNA Methylation Assay, Microarray, Expressing, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: Dissecting the MUC5AC/ANXA2 signaling axis: implications for brain metastasis in lung adenocarcinoma

doi: 10.1038/s12276-024-01255-6

Figure Lengend Snippet: a , b Total cellular fluorescence of cancer cells (scramble and MUC5AC KD cells) after coculture with astrocytes and microglia (1:2) respectively, for 48 h. c 2% agarose gel electrophoresis was performed to detect MUC5AC after treatment of A549 and A549-BrM cells with astrocyte-conditioned medium (ACM) d Cytokine array analysis of culture supernatants collected from cultures of human astrocytes (HAs) alone, A549-BrM cells alone, HAs + A549-BrM cells (2:1 ratio), and medium alone. HAs strongly secreted MIF, CCL2, IL-6, and IL-8. e 2% agarose gel electrophoresis showed a concentration (100–300 ng/ml)-dependent increase in MUC5AC expression compared to that in the control group. f The growth kinetics of A549-BrM cells after 100 ng/ml CCL2 treatment were compared to those of control cells. g Schematic showing the Sp1 binding site in the 5’-UTR of the MUC5AC gene. h ChIP-quantitative PCR analysis of Sp1 binding sites in the MUC5AC promoter. The data were presented as the fold enrichment values. i Western blot results showing the effect on MUC5AC and SP1 expression after treatment with an ERK inhibitor (PD98059) for 36 h. β-Actin was used as a loading control. P < 0.05 was considered to indicate statistical significance. Mean ± SD. Nonparametric t -test.

Article Snippet: The cells were then harvested by scraping, collected by centrifugation (3000 × g , 5 min, 4 °C), lysed, subjected to MNase digestion, and subjected to ChIP DNA enrichment with Sp1 (#9389, Cell Signaling Technology) and normal rabbit IgG.

Techniques: Fluorescence, Agarose Gel Electrophoresis, Concentration Assay, Expressing, Control, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancer cell

Article Title: Proteome instability is a therapeutic vulnerability in mismatch repair deficient cancer

doi: 10.1016/j.ccell.2020.01.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Nedd18 (Poly) , Cell Signaling , Cat# 2745; RRID: AB 10695300.

Techniques: Control, Ubiquitin Proteomics, Virus, Recombinant, Plasmid Preparation, Expressing, cDNA Synthesis, Extraction, DNA Extraction, Microarray, Sequencing, Mutagenesis, Protein Binding, Synthesized, Functional Assay, shRNA, Software

Journal: Cancer Cell

Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma

doi: 10.1016/j.ccell.2017.11.005

Figure Lengend Snippet:

Article Snippet: anti-cleaved Caspase-3 (Asp175) , Cell Signaling Technology , Cat#9661S; RRID: AB_2341188.

Techniques: Plasmid Preparation, Recombinant, Staining, Protease Inhibitor, Lysis, Mutagenesis, Gel Extraction, Microarray, DNA Methylation Assay, DNA Sequencing, shRNA, Control, Software

Journal: Cell reports

Article Title: Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK

doi: 10.1016/j.celrep.2019.09.070

Figure Lengend Snippet:

Article Snippet: Anti-AMPK-alpha , Cell Signaling Technology , Cat# 2532; RRID:AB_330331.

Techniques: Virus, Plasmid Preparation, Recombinant, Lysis, Saline, Modification, Isolation, Cell Culture, Membrane, Activity Assay, DNA Extraction, Glucose Assay, Bioassay, cDNA Synthesis, SYBR Green Assay, Western Blot, Microarray, Software

Journal: Cancer cell

Article Title: Proteome instability is a therapeutic vulnerability in mismatch repair deficient cancer

doi: 10.1016/j.ccell.2020.01.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HA (6E2) , Cell Signaling , Cat# 2367; RRID: AB 10691311.

Techniques: Control, Ubiquitin Proteomics, Virus, Recombinant, Plasmid Preparation, Expressing, cDNA Synthesis, Extraction, DNA Extraction, Microarray, Sequencing, Mutagenesis, Protein Binding, Synthesized, Functional Assay, shRNA, Software

Journal: Cancer Cell

Article Title: MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma

doi: 10.1016/j.ccell.2017.11.005

Figure Lengend Snippet:

Article Snippet: anti-phosphor-Histone H2A.X (Ser139) , Cell Signaling Technology , Cat#9718S; RRID: AB_2118009.

Techniques: Plasmid Preparation, Recombinant, Staining, Protease Inhibitor, Lysis, Mutagenesis, Gel Extraction, Microarray, DNA Methylation Assay, DNA Sequencing, shRNA, Control, Software