Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: a Proteome profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA) probed with serum-free-conditioned medium (SF-CM) of C33a-FL-CA IX or C33a-NS-CA IX cells incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h. Spot doublets of corresponding proteins with differential intensities are framed and indicated by arrows. Medium pooled from five independent experiments was used for the array. Spot intensities representing b pro-oncogenic basigin, and c anti-angiogenic thrombospondin-1 were analysed in culture medium samples of C33a-FL-CA IX versus C33a-NS-CA IX cells. Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD. d Validation of the differential levels of basigin and thrombospondin-1, respectively, by Western blotting analysis of medium precipitates (MP) and cell lysates (CL) of normoxic (NO) and hypoxic (HY) cells. (* P < 0.05, ** P < 0.01, *** P < 0.005).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Incubation, Biomarker Discovery, Western Blot

Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: Analysis of the differential expression of a IGFBP-2 and b IGFBP-3 in SF-CM samples from C33a-FL-CA IX and C33a-NS-CA IX incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h and evaluated by Proteome Profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA). Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD of five independent samples. c Western blotting analysis of CA IX, IGFBP-2 and IGFBP-3 protein levels in medium precipitates (MP) of C33a-FL-CA IX and C33a-NS-CA IX cells. Impairment of shedding in NS-CA IX is associated with increased IGFBP-2 and reduced IGFBP-3 secretion to culture medium. Quantitative PCR analysis of relative mRNA levels of d IGFBP-2, e IGFBP-3 and f IGF1 in C33a-FL-CA IX versus C33a-NS-CA IX cells normalised to β-actin mRNA. (*** P < 0.005, ns non-significant).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Quantitative Proteomics, Incubation, Western Blot, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling

doi: 10.7554/eLife.68348

Figure Lengend Snippet: ( A–L ) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or siCTRLsingle1; control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2). ( A,C,H ) Western blotting images of phospho-kinases spotted on the Proteome Profiler Human Phospho-Kinase Array in complete media (A) or under EGF stimulation (C, 50 ng/ml, 10 min) or serum-starved ( H ). Phospho-EGFR double spots are marked in red rectangles. ( B ) Immunoblot and quantification (n = 3 independent experiments) of p-EGFR (Y1068 or Y1086). p-EGFR levels were quantified by calculating the ratio between p-EGFR and EGFR, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( D, E ) Immunoblot and quantification (n = 3 independent experiments) of pSTAT3, p-ERK, and p-AKT. p-STAT3, p-ERK, and p-AKT levels were quantified by calculating the ratio between phospho-protein and total-protein, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( F ) Representative wide-field immunofluorescence images (left) and quantification (right) of pSTAT3 nuclear translocation corresponding to nuclei/cytoplasm mean intensity ratio of pSTAT3. ( G ) mRNA quantification of STAT3 target genes (bcl2, mmp2, mmp9 and hif2). ( I ) Quantification of EGFR mRNA level. ( J ) EGF secretion measured by ELISA. ( K ) mRNA quantification of EGFR ligands. ( L ) EGFR ligands secretion measured by ELISA. Scale bar = 10 µm. n ≥ 3 independent experiments; ns = non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ( F, I, J ) two-tailed t-test; ( B,C,E,G, K,L ) two-way ANOVA and Bonferroni’s multiple comparisons test; data are mean ± s.e.m. Figure 1—figure supplement 1—source data 1. Uncropped western blot for .

Article Snippet: Relative phosphorylation levels of 43 kinases and two related proteins were assessed using the Proteome Profiler Human Phospho-Kinase Array Kit (R and D Systems), according to the manufacturer’s instructions.

Techniques: Transfection, Control, Western Blot, Immunofluorescence, Translocation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International journal of oncology

Article Title: Oct4 suppresses IR‑induced premature senescence in breast cancer cells through STAT3- and NF‑κB-mediated IL‑24 production.

doi: 10.3892/ijo.2018.4391

Figure Lengend Snippet: Figure 8. Function of IL‑24 in Oct4‑mediated suppression of IR‑induced senescence. (A) Cytokine array of MCF7 cells. RT‑PCR analysis of MCF7 cells (B) cultured as indicated with the indicated primers. (C) Western blot analysis of adenoviral‑Luc- and adenoviral‑Oct4‑transduced MCF7 cells. RT‑PCR analysis of MCF7 cells (D) exposed to 4 Gy of IR, (E) treated with STAT3 inhibitor WP1066 (5 µM), (F) transfected with siRNA-targeting STAT3 or (G) treated with NF‑κB inhibitor Bay 11-7082 (10 and 20 µM) following transduction with adenoviral‑Luc or adenoviral‑Oct4. (H) Western blot analysis of adenoviral‑Luc- and adenoviral‑Oct4‑transduced MCF7 cells pretreated with Bay 11-7082 (10 µM) with the indicated antibodies. IL, interleukin; Oct4, octamer-binding transcription factor 4; RT‑PCR, reverse transcription-polymerase chain reaction; IR, ionizing radiation; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3; NF‑κB, nuclear factor κB; IκB, inhibitor of NF‑κB; SDF‑1α, stromal cell-derived factor 1α; GDF15, growth differentiation factor 15; MCP‑1, monocyte chemoattractant protein 1; CCL, CC chemokine ligand; PAI‑1, plasminogen activator 1; Rantes, regulated on activation, normal T-cell expressed and secreted; IP‑10, interferon γ-induced protein 10; CXCL10, CXC cytokine ligand 10; DKK1, dickkopf-related protein 10; msp, mammosphere; mono, monolayer- cultured; IRR, IR‑resistant; Luc, luciferase; siSTAT3, small interfering RNA against STAT3; sicon, control small interfering RNA; p-, phosphorylated.

Article Snippet: Cytokine array-base analysis was performed using a Human Cytokine Array kit (cat. no. ARY022; R&D Systems, Inc.), according to the manufacturer's protocol.

Techniques: Cell Culture, Western Blot, Transfection, Transduction, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA, Derivative Assay, Activation Assay, Luciferase, Control

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Caffeic acid selectively eliminates teratogenic human-induced pluripotent stem cells via apoptotic cell death.

doi: 10.1016/j.phymed.2022.154144

Figure Lengend Snippet: Fig. 3. Increase of caspase activities by CAA treatment in iPSCs. (A) Apoptosis-related proteins were detected by Western blotting in CAA-treated iPSCs and hDFs. Relative band intensities were calculated after normalization to β-actin expression. (B) iPSCs and hDFs were treated with 50 and 100 μM CAA for 24 h, and the caspase-3, −8, and −9 activities were determined. Relative caspase activity compared with CAA-untreated iPSCs and hDFs was expressed as the means ± SD (n = 3). (C) iPSCs were pre-treated with or without 10 μM of caspase-3 inhibitor (z-DEVD), caspase-8 inhibitor (z-IETD), caspase-9 inhibitor (z-LEHD), and pan-caspase inhibitor (z-VAD) for 30 min and then treated with 50 and 100 μM CAA. DMSO was treated as vehicle control. After 24 h, cells were stained with crystal violet solution and relative cell viability compared with CAA-untreated control iPSCs were expressed as the means ± SD (n = 3). **p < 0.01 vs. CAA-untreated control. ##p < 0.01 vs. Inhibitor-untreated control. Scale bar = 100 μM.

Article Snippet: The expression profile of human apoptosis-related proteins in CAA-treated or -untreated iPSCs was analyzed using a Proteome Profiler Human Apoptosis Array Kit A. Kim et al. Phytomedicine 102 (2022) 154144 (ARY009, R&D System, Minneapolis, MN, USA) according to the manufacturer’s instruction.

Techniques: Western Blot, Expressing, Activity Assay, Control, Staining

Journal: Biology

Article Title: Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

doi: 10.3390/biology11070980

Figure Lengend Snippet: Figure 6. Profiling of cytokines expressed by secretomes of irradiated or non-irradiated fibroblasts obtained with Proteome Profiler Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of dot blot for each protein normalized to positive spot pixels.

Article Snippet: Profili cytokines expres ed by secretomes of irradiated or non-irradi ted fibro lasts obtained with Proteome Profile Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of d t blot f r each protein normalized to positive spot pixels.

Techniques: Irradiation, Dot Blot

Journal: Biology

Article Title: Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

doi: 10.3390/biology11070980

Figure Lengend Snippet: Figure 8. Profiling of cytokines expressed by secretomes of irradiated untreated fibroblast or precon- ditioned with MSC-EV or MSC-CM obtained with Proteome Profiler Human XL cytokine Array Kit (R&D). (A) Representative image of dot blot; (B) graph showing signal density of dot blot of each protein normalized to positive spot pixels.

Article Snippet: Profili cytokines expres ed by secretomes of irradiated or non-irradi ted fibro lasts obtained with Proteome Profile Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of d t blot f r each protein normalized to positive spot pixels.

Techniques: Irradiation, Dot Blot

Journal: Biology

Article Title: Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

doi: 10.3390/biology11070980

Figure Lengend Snippet: Figure 9. MSC-EVs and MSC-CM protein composition. (A) Profiling of angiogenesis-regulating proteins secreted by MSC-EV or MSC-CM samples obtained with Proteome Profiler Human An- giogenesis Array Kit (R&D). At the top, representative image of dot blot and at the bottom, graph showing pixel density of dot blot of each protein normalized to positive spot pixels. (B) Profiling of cytokines secreted by MSC-EV or MSC-CM samples obtained with Proteome Profiler Human XL cytokine Array Kit (R&D). At the top, representative image of dot blot and at the bottom, graph showing pixel density of dot blot of each protein normalized to positive spot pixels (C): Quantification of IL8, MCP-1 and CXCL16 secreted by MSC-EV or MSC-CM samples by ELISA (mean ± SD), n = 3.

Article Snippet: Profili cytokines expres ed by secretomes of irradiated or non-irradi ted fibro lasts obtained with Proteome Profile Human XL cytokine Array Kit (R&D) (A) Representative image of dot blot, (B) graph showing signal density of d t blot f r each protein normalized to positive spot pixels.

Techniques: Dot Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Deoxyshikonin Mediates Heme Oxygenase-1 Induction and Apoptotic Response via p38 Signaling in Tongue Cancer Cell Lines

doi: 10.3390/ijms23137115

Figure Lengend Snippet: Profiling of DSK-induced apoptotic proteome in tongue cancer. ( A ) Representative images of array membranes corresponding to DSK-untreated and -treated protein expression in HSC-3 cells. Differentially expressed proteins are labeled and selected for further verification. ( B ) HSC-3 and SCC-9 cells were treated with various concentrations of DSK for 24 h and assessed for the expression of indicated apoptosis related proteins by immunoblotting. Densitometric analyses were quantified and normalized with internal controls (β-actin). The values represent the mean ± SD of three independent experiments. * p < 0.05 compared with untreated controls using Student’s t -test.

Article Snippet: Proteome of apoptotic tongue cancer cells in response to DSK treatment was analyzed by using a Proteome Profiler Human Apoptosis Array Kit (R&D Systems, Minneapolis, MN, USA), allowing to assess a panel of 35 key apoptotic proteins simultaneously.

Techniques: Expressing, Labeling, Western Blot

Journal: International journal of oncology

Article Title: Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells.

doi: 10.3892/ijo.2014.2397

Figure Lengend Snippet: Figure 3. Reduced production of VEGF and transcription of both VEGF-A and VEGF-B by C. militaris extract. (A) Dot blot-human angiogenesis array of MeWo culture medium after C. militaris extract or cordycepin treatment. Culture media of MeWo cells incubated with control, 5% C. militaris extract, 20 µM cordycepin, or 100 µM cordycepin for 48 h were used to detect the production of various angiogenesis-related proteins. All treatment groups showed lower production of angiogenin, TIMP-1, PDGF-AA, and VEGF compared to the control group. Production of TIMP-1 and VEGF was especially reduced in the 5% C. militaris treatment group. (B and C) qPCR of VEGF-A and VEGF-B. Transcription levels of VEGF-A and VEGF-B were quantified from MeWo cells after incubation with either 5% C. militaris extract (B) or 20 µM cordycepin (C) for 48 h. Transcription levels of VEGF-A and VEGF-B were significantly decreased after treatment with 5% C. militaris extract, while only VEGF-A was decreased after treatment with 20 µM cordycepin. *P<0.05 significantly different from the control group. ns, not significant.

Article Snippet: Screening for angiogenesis-related factors produced by MeWo cells was carried out by using the Human angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA).

Techniques: Dot Blot, Incubation, Control

Journal: International journal of oncology

Article Title: Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells.

doi: 10.3892/ijo.2014.2397

Figure Lengend Snippet: Figure 6. Treatment with C. militaris extract reduced angiogenesis. (A) Decrease of FITC-lectin intensity after treatment with 5% C. militaris extract. Using the Cultrex Directed In Vivo Angiogenesis Assay (DIVAA) (Trevigen, Gaithersburg, MD, USA), angioreactors containing a mixture of BME and DDW or BME and 5% C. militaris extract were implanted subcutaneously into the flanks of mice for 16 days. After extraction, FITC-lectin intensity was measured using excitation 485 nm, emission 510 nm. (B) Chorioallantoic membrane (CAM) assay showed that neovascularization was inhibited by C. militaris extract. Eight days post-fertilized chicken egg shells were cut to place silicon rings on the CAM. The inner spaces of the silicon rings were filled with DDW or 50 µl of C. militaris extract. Egg shells were covered using transparent tape, and eggs were incubated for 48 h. Results were observed by decrease or increase of vascular branch formation from main capillary vessels.

Article Snippet: Screening for angiogenesis-related factors produced by MeWo cells was carried out by using the Human angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vivo, Angiogenesis Assay, Extraction, Membrane, Chick Chorioallantoic Membrane Assay, Incubation

Journal: Frontiers in Oncology

Article Title: Mutant NPM1-Regulated FTO-Mediated m 6 A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis

doi: 10.3389/fonc.2022.817584

Figure Lengend Snippet: FTO Promotes Leukemic Cell Proliferation by Facilitating Cell Cycle and Suppressing Cell Apoptosis. (A) CCK-8 analysis of cell growth in the FTO-silenced OCI-AML3. (B) EdU analysis of cell proliferation in the FTO-silenced OCI-AML3 cells. The bar graph showed the percentage of EdU positive cells. Scale bar: 50 μm. (C) Flow cytometric analysis of cell cycle distribution in FTO-silenced OCI-AML3 cells. The bar graph showed the percentage of G0/G1, S, and G2/M phase cells. (D) Cell apoptosis was measured by flow cytometric analysis of the Annexin V/DAPI stained cells. The bar graph showed the cell apoptosis rate. (E) Western blot analysis of Cyclin A2, t-Caspase 9, and c-Caspase 9 protein level in the FTO-silenced OCI-AML3. β-actin was used as the internal control. The bar graph showed the relative level of protein. Data were presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell apoptosis was determined by an Annexin V-APC/DAPI Apoptosis Detection Kit (Elabscience, Wuhan, China, #E-CK-A258).

Techniques: CCK-8 Assay, Staining, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Mutant NPM1-Regulated FTO-Mediated m 6 A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis

doi: 10.3389/fonc.2022.817584

Figure Lengend Snippet: The Oncogenic Function of FTO is Dependent on m 6 A RNA Demethylase Activity. (A) CCK-8 analysis of cell proliferation in OCI-AML3 cells following transfection of the HA-FTO-WT and HA-FTO-MUT plasmids. (B) m 6 A dot blot analysis of the m 6 A level of global RNAs in OCI-AML3 cells with MA or DMSO treatment for 24 h. Methylene blue staining served as a loading control. (C) Dose-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 0, 10, 25, 50, and 100 μM MA for 24 h. (D) Time-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 100 μM MA for 0, 24, and 48 h. (E) EdU analysis of cell proliferation in OCI-AML3 cells upon 50 μM and 100 μM MA treatments of 24 h. The bar graph showed the percentage of EdU positive cells. Scale bar: 50 μm. (F) Flow cytometric analysis of the cell cycle distribution in OCI-AML3 cells upon 50 μM and 100 μM MA treatments of 24 h. The bar graph showed the percentage of G0/G1, S, and G2/M phase cells. (G) Cell apoptosis was measured by flow cytometric analysis of the Annexin V/DAPI stained cells. The bar graph showed the cell apoptosis rate. Data were presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell apoptosis was determined by an Annexin V-APC/DAPI Apoptosis Detection Kit (Elabscience, Wuhan, China, #E-CK-A258).

Techniques: Activity Assay, CCK-8 Assay, Transfection, Dot Blot, Staining, Control

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: GDF15 play a crucial role in the regulations of breast cancer cells activities by visfatin-treated ADSCs (vADSCs). ( A ) After the three-day co-culture of MDA-MB-231 cells and vADSCs (V50 and V100 group) or untreated ADSCs (Ctrl group), the CM was collected and analyzed by using a cytokine array kit. ( B ) The expression of GDF15 in the co-cultured CM was validated by ELISA represented in a histogram. The ADSCs and MDA-MB-231 cells collected from the co-culture system were extracted for cell lysate to analyze the GDF15 expression by western blotting. ( C ) The migration and invasion of MDA-MB-231 treated with GDF15 at various concentrations for 48 h were evaluated by using a transwell system. ( D ) The indirect co-culture was performed in the presence or absence of the GDF15 neutralizing antibody of for three days. After that, the migration and invasion of the MDA-MB-231 cells collected from the co-culture were evaluated in a transwell system. ( E ) The expression of phosphor-AKT (pAKT) of MDA-MB-231 treated with GDF15 (50 ng/mL) at different time point was detected by western blotting. ( F ) The pAKT was detected in the MDA-MB-231 cells from the co-culture by western blotting. ( G ) After the three-day co-culture in the presence or absence of the wortmannin (400 nM), the MDA-MB-231 cells were collected from the co-culture for performing the migration assay. All experiments were performed in triplicate.

Article Snippet: To explore the cytokine expression profile in the medium of the indirect co-culture of MDA-MB-231 and resistin-pretreated ADSCs, a Proteome profilerTM human XL cytokine array kit (ARY022, R&D Systems) was adopted.

Techniques: Co-Culture Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Migration