Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 1. Upregulation of ALDH1A3 in oxGBM cells is associated with the increased expression and release of pro-angiogenic factors PAI-1 and IL-8. GBM cell lines were transduced with ALDH1A3 for overexpression (oxGBM) or with empty vector (evGBM). (A) Confirmation of up-regulation of ALDH1A3 mRNA level in oxGBM cells by RT2-PCR. (B) Confirmation of up-regulation of ALDH1A3 protein expression by Western blot. wt, wild-type cells. The uncropped blots are shown in Figure S7; (C) Angiogenesis array. The blots showed duplicated dots for 55 angiogenesis-related proteins in the media of oxU373 or evU373. 10 of 55 proteins were upregulated more than 2-fold in ox group compared to ev group (indicated by rectangle). They are: (1) Ang-1, (2) artemin, (3) TF, (4) ET-1, (5) GM-CSF, (6) IL-8, (7) PDGF-AA, (8) PAI-1, (9) PEDF, and (10) uPA. (D) Semi-quantification of the dots representing PAI-1 and IL-8. (E) Immunofluorescence staining of GBM cells. U373 (left panel) and LN229 (right panel). Co-localization of ALDH1A3 with PAI-1 and IL-8 was observed in oxGBM cells, whereas no immunoreactivity of PAI-1 and IL-8 was detected in evGBM cells. (F) mRNA expression of PAI-1 and IL-8 in transduced U373 cells and the effect of inhibitors. Tiplaxtinin (Tip, 30 µM) and reparixin (Rep, 1 µM) are the specific inhibitors of PAI-1 and IL-8 receptors CXCR1/2, respectively. (G) Detection of PAI-1 and potential signaling proteins by Western blot. IOD: optical density. The uncropped blots are shown in Figure S8. **, p < 0.01; ***, p < 0.001, compared with ev. ##, p < 0.01; ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 2. Overexpression of ALDH1A3 in GBM cells activated endothelial angiogenesis in indirect co-culture with endothelial cells (ECs), which was reversed by treatment with respective inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Indirect co-culture was performed by culture of HBMECs in a conditioned medium (CM) containing the media derived from evGBMs or oxGBMs and ECGM in a ratio of 1:1. PAI-1 inhibitor tiplaxtinin (Tip, 30 µM) and CXCR1/2 inhibitor reparixin (Rep, 1 µM) or vehicle DMSO (0.1%) was added to CM, followed by EC behavior study. All data were reproduced in three independent experiments. (A) Proliferation assay in HBMEC and HUVEC. Indirect co-culture of HBMECs and HUVECs with CM derived from oxU373 and oxLN229 stimulated EC proliferation, which was completely reversed by the treatment of tiplaxtinin, not by reparixin. (B) Scratch assay in HBMEC. Left panel: images were acquired 24 h after scratching. Scale bar: 200 µm. Right panel: quantitative analysis. Culture of HBMECs with CM derived from oxU373 or oxLN229 (oxCM) significantly promoted HBMEC migration, which was reversed by the treatment of tiplaxtinin and reparixin, respectively. (C) Transwell invasion assay in HBMEC. Left panel: Representative images of invaded cells were acquired after 24 h of incubation. Scale bar: 100 µm. Right panel: quantitative analysis. Culture of HBMECs with oxCM accelerated HBMEC invasion. This effect was significantly inhibited by the treatment of reparixin but not by tiplaxtinin. (D) Tube formation assay in HBMEC. Left panel: representative images of tube formation. Scale bar: 200 µm. Right panel: quantitative analysis of branching points per field. Tube formation in HBMECs was stimulated by incubation with oxCM, which was completely diminished by both inhibitors. (E) Sprouting assay in HBMEC. Left panel: representative images of sprouting in HBMECs after 24 h of co-culture. Scale bar: 100 µm. A pronounced increase in sprouting was observed in HBMECs cultured in oxCM. Tiplaxtinin and reparixin suppressed the sprouting effect resulting from oxCM. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with evCM. #, p < 0.05; ##, p < 0.01 and ###, p < 0.001, compared with oxCM.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Over Expression, Co-Culture Assay, Derivative Assay, Proliferation Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Incubation, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 4. oxGBM-derived culture media stimulated angiogenesis on CAM, which was fully rescued by the treatment of tiplaxtinin and reparixin. CAM was incubated with the culture media derived from evU373 or oxU373 cells with or without tiplaxtinin (Tip, 30 µM) or reparixin (Rep, 1 µM) or DMSO (as a vehicle control, 0.1%) for 72 h. (A) Microscopy view of the vasculature structure on CAM. More enriched microvessel network was clearly visible in the ox group, which was significantly reduced in tiplaxtinin- and reparixin-treated CAMs. The images were acquired using a stereo microscope on ED13 (scale bar: 1 mm). Stem vessel (arrow); branched microvessel network (arrow heads). (B) Histological features of CAM after hematoxylin-eosin (H&E) staining. The CAM consists of the chorionic epithelium layer (ChE), allantoic epithelium (AE) layer, and the mesenchymal (MES) layer (arrows). Microvessel (arrowheads) density was much higher in the MES layer of ox section compared to ev section, which was clearly reduced in tiplaxtinin- and reparixin-treated sections. Scale bar: 50 µm. (C) Quantitative analysis of branching point of vessels based on microscopy images. The number of branching points and microvessels was counted by the ImageJ software (v1.1.53t) in 3 fields/CAM (n = 10 CAM/group). (D) Quantitative analysis of microvessel numbers based on H&E-stained CAM sections. Microvessel number was counted manually on H&E-stained CAMs. 10 fields/section (n = 6 sections/group). ***, p < 0.001, compared with ev. ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Incubation, Control, Microscopy, Staining, Software

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