Journal: BMC cancer

Article Title: Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer.

doi: 10.1186/1471-2407-13-518

Figure Lengend Snippet: Figure 5 EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signal- ling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

Article Snippet: Whole cell lysate of EGF-treated A431 epithelial carcinoma cells used as positive control was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Positive Control, Control, Software

Journal: Bioconjugate chemistry

Article Title: Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

doi: 10.1021/acs.bioconjchem.5b00613

Figure Lengend Snippet: Fig. 6. Detection of proteins in cell lysates. A panel of 24-plex antibody binders was used to detect their respective target proteins in MCF7, K562, A549, and A431 cell lysates. The cell lysates were diluted to different concentrations and reacted with a mixture of antibody binders. Only selected examples of protein targets CSTB (A), CASP3 (B), Ki-67(C), and GATA3 (D) are shown. Ct values were shown as the means ± SD. Ct: cycle threshold.

Article Snippet: MCF7, K562, A549, and A431 cell lysates were obtained from Novus (Littleton, CO).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes

doi: 10.3390/ijms24043896

Figure Lengend Snippet: Ox-mtDNA activates pyroptosis. ( A ) Log10-transformed, glucose-adjusted ox-mtDNA levels from Low Risk (LR) MDS PB (n = 100) and BM plasma (n = 70), and Normal PB (n = 30). ( B ) Western blot of U937 and SKM1 cells treated with 10-fold increasing isolated mtDNA of the ND1 gene region amplified with oxidized guanosine (ox-mtDNA) for 2 h to induce Caspase-1 (arrow depicts cleaved fragment) and phosphorylated NFκB to establish dosage (representative blot of n = 3). Remaining figures, ox-mtDNA treatment is 50 ng/mL ox-mtDNA for 2 h unless otherwise stated. ( C ) Western blot of SKM1 and U937 cells treated with ox-mtDNA showing, cleavage of caspase-1 (arrow depicts cleaved fragment), and IL-1β (arrow depicts cleaved fragment) demonstrating inflammasome activation (representative blot of n = 3). ( D ) Fold change of caspase-1 activity quantified by Caspase-1 Glo ® assay in SKM1 and U937 cells treated with ox-mtDNA, (mean ± SEM of n = 5). ( E ) Fold change of LDH media release (measurement of cell death), quantified by LDH-Glo™ Cytotoxicity Assay, of SKM1 and U937 cells treated with ox-mtDNA (mean ± SEM of n = 5). ( F ) Fold change of caspase-1 activity quantified by Caspase-1 Glo ® assay in SKM1 cells pretreated with either CRISPR KO (pooled guides) for NLRP3 or 10 uM MCC950 for 48 h prior to treatment with ox-mtDNA (mean ± SEM of n = 4). ( G ) Representative confocal IF micrographs showing increased ASC specks in ox-mtDNA stimulated cells compared to untreated controls [DAPI (blue), ASC (green) (×2520)]. ( H ) Quantification of 1-to-2 μm ASC speck IF, at least 200 cells counted per group, (mean ± SEM of n = 3). ( I ) Immunoblot of ASC following chemical crosslinking, cells treated with ox-mtDNA or positive control LAN (LPS + ATP + Nigericin), arrow indicates oligomers. ( J ) Western Blot for PARP and Caspase-3. Western blot of SKM1 and U937 cells treated with ox-mtDNA, and an apoptosis positive control (A431 EGF Stimulated) showing induction of PARP1, cleavage of caspase-3 (arrow depicts cleaved fragments, representative blots of n = 3). ( K ) Fold change of caspase-1 activity quantified by Caspase-1 Glo ® assay and LDH media release quantified by LDH-Glo™ Cytotoxicity Assay in primary Normal BM-MNC cells treated with ox-mtDNA (mean ± SEM of n = 3). ( L ) Colony formation assay for hematopoiesis in Healthy BM-MNCs treated with 50 ng/mL ox-mtDNA for 14 days (representative picture). ( M ) Quantification of various hematopoietic progenitor colonies in response to treatment with 50 ng/mL of ox-mtDNA for 14 days (mean ± SEM of n = 4). In this figure, significance was assessed by either paired t -test (comparison between two groups only) or ordinary one-way ANOVA with multiple comparison analysis in GraphPad Prism. p values are shown as asterisk: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Apoptosis positive control was A431 Whole Cell Lysate EGF Stimulated (Rockland Immunochemicals, Inc., Limerick, PA, USA).

Techniques: Transformation Assay, Clinical Proteomics, Western Blot, Isolation, Amplification, Activation Assay, Activity Assay, Glo Assay, Cytotoxicity Assay, CRISPR, Positive Control, Colony Assay, Comparison

Journal: Hepatology (Baltimore, Md.)

Article Title: Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats.

doi: 10.1053/jhep.2002.31355

Figure Lengend Snippet: Fig. 3. Group phenotyping of LPC strains and clonal lines cultured on STO feeder layers. Cellular extracts from 22 sources were made 3 days postplating and analyzed (lanes 4-25) for AFP, albumin, CK14, c-kit, CD34, and desmin by Western blotting. The names of individual cell strains and cell lines are shown above each lane; the larger single numbers above the names designate group assignments (see Table 2). Extracts used as internal controls were obtained from neonatal liver (BRL) and, as indicated, from bone marrow and skeletal muscle (lane 1), STO fibroblasts (lane 2), freshly isolated adult rat hepatocytes and, as annotated, human A431 cells (lane 3). The 6 positive control markers included AFP, albumin, CK14, and c-kit (BRL), CD34 (bone marrow), and desmin (skeletal muscle) in lane 1; and CK14 (A431 cells) and albumin (adult hepatocytes) in lane 3. Negative controls were provided by STO cells for all markers (lane 2), by adult rat hepatocytes for AFP and c-kit (lane 3), and for CK14 (not shown). Molecular weights of the 6 markers are listed to the right of lane 25.

Article Snippet: The following materials were obtained commercially: dexamethasone and bFGF (Sigma); Oncostatin M (R&D Systems, Minneapolis, MN); Matrigel (Collaborative Research, Bedford, MA); human epidermoid carcinoma cell line A431 lysates (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Cell Culture, Western Blot, Isolation, Positive Control

Journal: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: Inhibitory effects of AQP3 and PLD2 siRNA sequences on the target gene. siRNA transfection and reverse transcription-quantitative polymerase chain reaction analyses were performed. The groups were as follows: CK group, normal cultured A431 cells; NC group, A431 cells were transfected with NC siRNA; 1, AQP3-homo-612 siRNA group; 2, AQP3-homo-363 siRNA group; 3, AQP3-homo-360 group; 4, PLD2-homo-602 group; 5, PLD2-homo-1352 group; 6, PLD2-homo-5338 group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Culture, Small Interfering RNA, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: Inhibitory effect of siRNA transfection on the proliferation of A431 cells. Cell proliferation was analyzed and the results are representative of three independent experiments. The statistical significance between two groups was determined using a paired-samples t-test. **P<0.01, compared with the NC group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Small Interfering RNA, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: AQP3 siRNA and PLD2 siRNA increase the apoptosis of A431 cells. Cell apoptosis was determined; results are representative of three independent experiments. The statistical significance between two groups was determined using a paired-samples t-test. **P<0.01, compared with the NC group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Small Interfering RNA, Control, Negative Control