Journal: Developmental cell

Article Title: Mammalian SEPT2 is required for scaffolding nonmuscle myosin II and its kinases.

doi: 10.1016/j.devcel.2007.09.001

Figure Lengend Snippet: Figure 4. Dissociation of SEPT2 from Myosin II Alters Myosin II Activity and Increases the Number of Binucleated Cells (A) Representative western blots of CHO-K1 whole-cell lysates prepared after incubating cells with PBS, the TAT-CCT3-pt, or TAT-CCT5 for 1.5 hr. Each lysate was probed with antibodies against the indicated proteins. (B) Quantification of the western blots showing relative levels of myosin II, MYPT1, pMYPT1, SEPT2, SEPT9, MLC, pMLC, and ppMLC. Each exper- iment was done a minimum of three times, normalized to PBS transduction. The error bars reflect the standard deviation. *p < 0.05. (C) Representative western blots of CHO-K1 whole-cell lysates prepared after transfecting with nonspecific shRNAi (shRNAi Scramble) or shRNAi silencing SEPT2. Each lysate was probed with antibodies against the indicated proteins. (D) A representative recording of cell division shown over time depicting cells that were transduced with the TAT-CCT5 or TAT-CCT3-pt. Recordings were started at the beginning of karyokinesis. The number on each panel indicates the time elapsed from the starting point, expressed in minutes.

Article Snippet: Collected samples were probed for SEPT9 (1:1000; Surka et al., 2002), SEPT2 (1:1000), myosin II (1:1000), MYPT1 or pMYPT1 (1:1000; Upstate Biotechnology), and pMLC or ppMLC (1:1000; Cell Signaling Technology).

Techniques: Activity Assay, Western Blot, Transduction, Standard Deviation

Journal: Clinical Cancer Research

Article Title: Spatial Heterogeneity, Stromal Phenotypes, and Therapeutic Vulnerabilities in Colorectal Cancer Peritoneal Metastasis

doi: 10.1158/1078-0432.CCR-24-3780

Figure Lengend Snippet: Development of A5, a novel PAI-1–neutralizing antibody. A, Graphical illustration of phage screen and identification of PAI-1–neutralizing antibody. B, Recognition of different conformations of recombinant PAI-1 by A5 in Western blotting. C, A5 neutralized PAI-1 function in inhibiting tPA and uPA. D, Competitive binding of A5 with fluorine-containing tiplaxtinin on stable active PAI-1 assessed by 19F-NMR. E, Inhibition of proliferation of PM cells treated with PAI-1–positive ascites in vitro by 150 µg/mL A5 in biological triplicates. Human IgG served as a negative control. F, STAT3 signaling activation upon the exposure to PM ascites was suppressed by A5 as compared with IgG control. G, GSEA comparisons of HALLMARK pathway changes between CRC cells treated with A5 ( n = 2) vs. IgG ( n = 2). Pathways with an FDR adjusted P value of less than 0.05 are opacified. CRC, colorectal cancer; NES, normalized enrichment score.

Article Snippet: Then 25 μg of the protein lysate was used to measure total STAT3 and p-STAT3 (Tyr705) by ELISA using the PathScan Total Stat3 Sandwich ELISA kit (Cell Signaling Technology, 7305C) and the PathScan Phospho-Stat3 (Tyr705) Sandwich ELISA kit (Cell Signaling Technology, 7300C), respectively.

Techniques: Recombinant, Western Blot, Binding Assay, Inhibition, In Vitro, Negative Control, Activation Assay, Control

Journal: Acta pharmaceutica (Zagreb, Croatia)

Article Title: Insights into the mechanism of antiproliferative effects of primaquine-cinnamic acid conjugates on MCF-7 cells.

doi: 10.2478/acph-2018-0021

Figure Lengend Snippet: Fig. 5. The effects of test compounds on the levels of apoptosis signaling proteins. Results represent mean values ± SEM. Significant difference vs. control, *p < 0.05.

Article Snippet: Apoptosis assay: Apoptosis protein levels Apoptosis proteins were measured using a PathScan Apoptosis Multi-Target Sandwich ELISA Kit (Cell Signaling Technology, USA) according to the manufacturer’s instructions.

Techniques: Control

Journal: Scientific Reports

Article Title: IL-1α is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity

doi: 10.1038/srep14756

Figure Lengend Snippet: ( a ) Human HT1080 fibrosarcoma and HaCaT human keratinocytes were subjected to genotoxic stresses: UV irradiation (5 mJ/cm 2 ), 10 mM H 2 O 2 or 50 μgr/ml Bleomycin. 16 h post exposure, hIL-1α ELISA was used to measure secreted IL-1α in cell supernatants. All experiments were performed in triplicates and data are expressed as mean ± SD. ( b ) Nuclear/cytoplasmic re-localization of IL-1α after DNA damage. Live cell imaging of B16 melanoma cells expressing GFP-IL-1α during treatment with 100 μ M H 2 O 2 . Images were collected every 30 min for a period of 24 h. Representative images from indicated time points are shown (for full video see , for averaged fluorescence intensities see also ) White scale bars, 20 μ m. ( c , d ) Nuclear IL-1α co-localizes with γH2AX foci after genotoxic stress. ( c ) B16 melanoma cells expressing GFP-IL-1α were treated with Etoposide 10 µg/mL for 2 h or ( d ) microirradiated with femtosecond laser pulses at λ = 775 nm (see also and ). After fixation of cells, detection of GFP-IL-1α, DAPI or immunostaining of γH2AX was preformed and visualized by confocal microscopy. White scale bars, 20 μ m ( e ) Recruitment kinetics of IL-1α to DNA damage sites. B16 melanoma cells expressing IL-1α–GFP were laser-microirradiated along a single line to induce DNA damage. Fluorescence intensity in the damaged region was measured up to 15 min from irradiaton in 1 min intervals. Data is expressed as mean ± SEM of increase in fluorescence intensity (n = 10 cells). ( f ) IL-1α localizes to Cyclobutane Pyrimidine Dimers (CPD) induced via laser microirradiation. B16 melanoma cells expressing GFP-IL-1α were laser irradiated and CPDs were visualized by immunostaining using specific antibodies. White scale bars, 20 μ m.

Article Snippet: The cells were allowed to express GFP-IL-1α and left to recover for an additional 24 h. To induce DNA damage, UV was applied as described above and levels of secreted GFP-IL-1α were measured by measuring GFP using the PathScan total GFP Sandwich ELISA kit (Cell signaling).

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Expressing, Fluorescence, Immunostaining, Confocal Microscopy

Journal: Scientific Reports

Article Title: IL-1α is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity

doi: 10.1038/srep14756

Figure Lengend Snippet: ( a ) IL-1α precursor is recognized by a pan acetyl antibody. Endogenous IL-1α was immunoprecipitated (IP) from nuclear extracts of Raw 264.7 cells, either induced or non-induced with 100 ng/ml LPS. Total IP proteins were separated over 15% SDS PAGE, transferred to nitrocellulose membranes and blotted with anti-mouse IL-1α (top panel) or anti-Kac (bottom panel). Acetylated IL-1α is marked by arrows and IP antibody light and heavy chain signals are indicated. ( b ) Annotated MS/MS spectrum of the tryptic peptide VTVSATSSN(Deam)GK(Acetyl)ILK (MH2 + 724.40 Da) showing acetylation of IL-1α (Uniprot ID P01582) at K82 and N80 deamidation. ( c ) PrecIL-1α K82 mutants affect IL-1α sub-cellular localization. Confocal microscopic analysis of GFP tagged WT IL-1α and mutations of precIL-1α K82 to glutamine (precIL-1α K82Q, mimicking acetylation) and to arginine (precIL-1α K82R non-acetylateable). White scale bars, 20 μ m ( d ) IL-1α K82 mutations reduce cytokine secretion after DNA damage. Mouse B16 cells were transfected with the indicated GFP IL-1α plasmids. The cells were then subjected to 100 μM H 2 O 2. 16h after stress induction levels of secreted GFP IL-1α in cell growth medium was measured using a GFP ELISA. GFP IL-1α levels in cell lysates were used to normalize for transfection efficiencies and non-transfected cells were used as negative controls. Data are expressed as mean ± SD of three independent experiments. ( e ) Histone deacetylase inhibition by TSA increases IL-1α nuclear localization. Images of cells expressing GFP IL-1α either non-treated (control) or treated with TSA (100 ng/ml) were collected every hour for 22 h and representative images for three time points (0, 11 and 22 hours) are shown (For averaged fluorescence intensities of nuclear/cytoplasmic ratios see ). ( f ) HDAC-1 and IL-1α can co-localize at DNA damage lesions. Cells expressing GFP IL-1α were laser-microirradiated for the induction of DNA damage. Localization of HDAC-1 and IL-1α–GFP were visualized by confocal microscopic analysis.

Article Snippet: The cells were allowed to express GFP-IL-1α and left to recover for an additional 24 h. To induce DNA damage, UV was applied as described above and levels of secreted GFP-IL-1α were measured by measuring GFP using the PathScan total GFP Sandwich ELISA kit (Cell signaling).

Techniques: Immunoprecipitation, SDS Page, Tandem Mass Spectroscopy, Transfection, Enzyme-linked Immunosorbent Assay, Histone Deacetylase Assay, Inhibition, Expressing, Control, Fluorescence

Journal: Cancer biology & therapy

Article Title: PDGFRbeta and HIF-1alpha inhibition with imatinib and radioimmunotherapy of experimental prostate cancer.

doi: 10.4161/cbt.6.11.4854

Figure Lengend Snippet: Figure 3. PDGFRb and PDGF‑BB expression in PC‑3 xenografts. (A) PDGF‑BB measurements in lysates prepared form PC‑3 xenografts extirpated from untreated control mice. Tumor lysates were analyzed for the presence of human and mouse PDGF‑BB using the ELISA kit. (B) Phosphorylation of PDGFRb in PC‑3 tumor and in mouse organs. Liver, kidney, heart and tumor were removed from four mice and lysates were prepared. Phosphorylation levels were measured using the commercial phospho‑PDGFRb (Try751) sand‑ wich ELISA kit. (C) Inhibition of insulin‑like growth factor‑I (IGF‑1) in PC‑3 xenografts extirpated from mice treated either with one IP dose of imatinib or one IP dose of PBS.

Article Snippet: The effect of imatinib on in vivo and in vitro PDGFRb phosphorylation levels was determined using the PathScan phospho-PDGFRb sandwich ELISA kit (Cell Signaling Technology, Inc., Beverly, MA).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Inhibition

Journal: Cell Death Discovery

Article Title: Counteracting gemcitabine+nab-paclitaxel induced dysbiosis in KRAS wild type and KRAS G12D mutated pancreatic cancer in vivo model

doi: 10.1038/s41420-023-01397-y

Figure Lengend Snippet: Representative immunohistochemical analyses of Ki67 (×20 magnification), Phospho- γH2A.X (×20 magnification), Arginase 1 (×20 magnification), and AIF (×10 magnification) in BxPC-3 ( A ) and PANC-1 ( B ) tumor sections. Scale bar 50 µm.

Article Snippet: Antigen retrieval was carried out using preheated target retrieval solution for 30 min. Tissue sections were blocked with FBS serum in PBS for 60 min and incubated overnight with the following primary antibodies: Ki67 (Thermoscientific, Italy, MA5-14520), Phospho-γH2A.X (Abcam, Italy, ab1174), Arginase-1 (GeneTex, USA, California, GTX109242), Apoptosis-Inducing Factor or AIF (Cell Signaling, USA, Massachusetts, 5318), Myeloperoxidase (Abcam, ab208670), CD41 (Abcam, ab181582), PAX5 (Abcam, ab109443), TER119 (Abcam, ab91113), and Endomucin (Abcam, ab106100).

Techniques: Immunohistochemical staining

Journal: Cell Death Discovery

Article Title: Counteracting gemcitabine+nab-paclitaxel induced dysbiosis in KRAS wild type and KRAS G12D mutated pancreatic cancer in vivo model

doi: 10.1038/s41420-023-01397-y

Figure Lengend Snippet: Representative pictures of hematoxylin/eosin, Alcian Blue/PAS and Ki67 in BxPC-3 ( A ) and PANC-1 ( B ) small intestine sections (×20 magnification). Scale bar 50 µm.

Article Snippet: Antigen retrieval was carried out using preheated target retrieval solution for 30 min. Tissue sections were blocked with FBS serum in PBS for 60 min and incubated overnight with the following primary antibodies: Ki67 (Thermoscientific, Italy, MA5-14520), Phospho-γH2A.X (Abcam, Italy, ab1174), Arginase-1 (GeneTex, USA, California, GTX109242), Apoptosis-Inducing Factor or AIF (Cell Signaling, USA, Massachusetts, 5318), Myeloperoxidase (Abcam, ab208670), CD41 (Abcam, ab181582), PAX5 (Abcam, ab109443), TER119 (Abcam, ab91113), and Endomucin (Abcam, ab106100).

Techniques: