Journal: Med (New York, N.Y.)

Article Title: Local production of lactate, ribose phosphate, and amino acids within human triple-negative breast cancer

doi: 10.1016/j.medj.2021.03.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MOLT-4 (Santa Cruz Biotechnology sc-2233, acute lymphoblastic leukemia), MCF7+EGF+β-estradiol (Santa Cruz Biotechnology sc-24730, breast adenocarinoma), and Jurkat+Calyculin (Santa Cruz Biotechnology sc-2277, human acute T cell leukemia induced with Calyculin A) cell lysates were printed on each array as quality control samples.

Techniques: Plasmid Preparation, Recombinant, Software

Journal: International Journal of Molecular Medicine

Article Title: Novel effect of estrogen on RANK and c-fms expression in RAW 264.7 cells

doi: 10.3892/ijmm.20.1.97

Figure Lengend Snippet: Figure 1. (Left panels) Estrogen receptors ER α and ß RNA expression in macrophage cells. RAW 264.7 cells were analyzed by RT-PCR for the presence of the mRNA encoding ER α and ß. Cells were cultured under standard growth conditions, and RNA was extracted and retrotranscribed using the reverse transcriptase enzyme. ER α and ß were expressed as specific single bands. (Right panels) Western blot analysis for ERs. The whole cell lysates from RAW 264.7 cells were analyzed for the expression of ER α and ß. The positive controls were MCF7 whole cell lysate for α and NIH/3T3 whole cell lysate for ß. Cont, control.

Article Snippet: Controls for both α and ß were used (MCF7 whole cell lysate for α and NIH/3T3 whole cell lysate for ß, Santa Cruz Biotechnology, Inc., CA).

Techniques: RNA Expression, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Reverse Transcription, Western Blot, Expressing, Control

Journal: ACS Omega

Article Title: Mechanical Transfer of Black Phosphorus on a Silk Fibroin Substrate: A Viable Method for Photoresponsive and Printable Biomaterials

doi: 10.1021/acsomega.3c09461

Figure Lengend Snippet: (a) Viability of MCF7 cancer cells after 24 h of incubation with PBS 1× (CTR), SF, SF/BP 0.03 mg, SF/BP 0.056 mg, and BP 0.056 mg reported without irradiation (in gray), irradiation with a 110 kJ/m 2 dose for 220 s (in blue), and irradiation with a 220 kJ/m 2 dose for 440 s (in orange). (b) AO–DAPI double staining was performed for each condition, and viability (%) was reported in each image.

Article Snippet: MCF7 cells were used as a well-characterized breast cancer cell line and were purchased from Elabscience Biotechnology (Houston, Texas).

Techniques: Incubation, Irradiation, Double Staining

Journal: Oncogene

Article Title: Breast cancer dissemination promoted by a neuregulin-collagenase 3 signalling node.

doi: 10.1038/onc.2015.337

Figure Lengend Snippet: Figure 2. NRG expression favors tumor growth and spreading. (a) In vitro analysis of the effect of NRGα2c expression on cell proliferation. A total of 25 000 MCF7 or MCF7-NRGα2c cells were plated in each well and cell proliferation measured by MTT metabolization. Results are plotted as the raw absorbance values, and represent the mean ± s.d. of quadruplicates. (b) Signalling status of the NRG-ErbB pathway in MCF7 or MCF7-NRGα2c cells. Analyses of the different signaling intermediates was performed by western blotting of cell extracts prepared from luciferized MCF7 or MCF7-NRGα2c cells. The values at the bottom represent luminescent readings (RLU) from both cell lines. (c) Superior invasiveness properties of MCF7 cells expressing NRGα2c as compared with wild-type MCF7 cells. A total of 20 000 cells were plated on top of a matrigel cushion lied on transwells. Culture media was added at the bottom of the well. In the case of MCF7 cells, the culture media of some samples included NRG (10 nM, column 2). Forty-eight hours later, invading cells were identified by crystal violet staining and counted. Results are plotted as the mean ± s.d. of quadruplicates. (d) Tumor mass analyses of mice injected with MCF7 or MCF7-NRGα2c cells. Photon fluxes at 60 and 90 days were taken using an IVIS50 apparatus, and data quantitated using the Igor Pro 4.0 software (Xenogen). (e) Luminescence analyses of mice injected with MCF7-Luc or MCF7-NRGα2c-Luc cells. Pictures were taken 90 days after injection. The rainbow bar at the right of each image represents the intensity scale of the luminescence signal. The arrow points to a disseminated lesion. The table at the bottom indicates the number of mice with metastasis of a total of six mice used in each of the analyzed groups.

Article Snippet: Preparation of cell lysates for immunoprecipitation or western analyses was as described.39,40 For the antibody arrays, 600 μg of MCF7 cell lysates treated or not with NRG were hybridized to ARY003B antibody arrays (R&D Systems, Abingdon, UK).

Techniques: Expressing, In Vitro, Western Blot, Staining, Injection, Software

Journal: Oncogene

Article Title: Breast cancer dissemination promoted by a neuregulin-collagenase 3 signalling node.

doi: 10.1038/onc.2015.337

Figure Lengend Snippet: Figure 3. Major gene categories deregulated by NRG in MCF7 cells. (a) qPCR analyses of genes included in major pathways deregulated by NRG. MCF7 cells were treated with NRG (10 nM) for different times (3, 6, 12, 24 h, shown only the 24 h time-point) and expression analyses performed. Changes in expression with respect to the resting level (basal = 1) are plotted. (b) Western blot analyses of the proteins affected by NRG treatment of MCF7 cells. MCF7 cells were treated with NRG (10 nM) for the indicated times. Fifty micrograms of protein extracts were loaded in each lane, and blots probed with antibodies against the indicated antigens. (c) Time-course analyses of the effect of NRG on the expression of collagenase 3. Exponentially growing MCF7 cells were washed twice with serum-free culture media and treated with NRG at different times. Culture media were collected, and cells were lysed. Collagenase 3 levels in cells were analyzed by western blotting using 50 μg of the cell lysates and the collagenase 3 antibody. Collagenase 3 in media samples was analyzed after immunoprecipitation and western blotting with the same antibody. (d) Release of active collagenase 3 by MCF7 cells upon treatment with NRG. Cells were treated with 10 nM NRG for the indicated times, and collagenase 3 activity in the culture media analyzed using a fluorescent enzymatic assay. Results are shown as the mean ± s.d. of triplicates. P indicates significance values with respect to resting measurements of activity. (e) MCF7 or T47D cells were serum-starved for 12 h and then treated for 6 h with 10 nM NRG. Analyses of collagenase 3 and GAPDH expression were directly performed on cell extracts, while that HER2 and pHER2 were carried out after immunoprecipitation using anti-HER2 antibodies.

Article Snippet: Preparation of cell lysates for immunoprecipitation or western analyses was as described.39,40 For the antibody arrays, 600 μg of MCF7 cell lysates treated or not with NRG were hybridized to ARY003B antibody arrays (R&D Systems, Abingdon, UK).

Techniques: Expressing, Western Blot, Immunoprecipitation, Activity Assay, Enzymatic Assay

Journal: Oncogene

Article Title: Breast cancer dissemination promoted by a neuregulin-collagenase 3 signalling node.

doi: 10.1038/onc.2015.337

Figure Lengend Snippet: Figure 4. Collagenase 3 mediates NRG-induced dissemination of breast cancer cells. (a–c) Effect of collagenase 3 knockdown on NRG-induced invasion (a), adhesion (b) and wound healing (c). MCF7 cells transfected with a control short hairpin or a short hairpin against collagenase 3 were treated with or without 10 nM NRG. The number of invading cells (a) was measured 3 days later. In (b), cells were plated on fibronectin and cell adhesion was analyzed 2 h later. Representative images of adherent cells (b) or wound-healing experiments (c) are shown at the right. During wound-healing experiments cultures were treated with mitomycin C (1 μm). Scale bar in (b) and (c) = 100 μm. Results are shown as the mean ± s.d. of quadruplicates. Western analyses of collagenase 3 and GAPDH levels in MCF7 cells transfected with a control short hairpin or a short hairpin against collagenase 3 are shown. (d–f) Xenograft mice model and analysis of tumor width in mice injected with 5 × 106 MCF-7α2c-sh-control or MCF-7α2c sh-collagenase 3 cells. Images of tumoral masses in both types of xenografts are shown in (d), as well as indication of the number of mice with metastasis in each experimental condition. Tumor width was measured using the Living Image software and compared (day 90) using two-sided Student’s t-test. Mean values ± s.d. are shown (e). (f) Analysis of collagenase 3 in tumor xenograft samples. GAPDH was used as a loading control.

Article Snippet: Preparation of cell lysates for immunoprecipitation or western analyses was as described.39,40 For the antibody arrays, 600 μg of MCF7 cell lysates treated or not with NRG were hybridized to ARY003B antibody arrays (R&D Systems, Abingdon, UK).

Techniques: Knockdown, Transfection, Control, Western Blot, Injection, Software

Journal: Oncogene

Article Title: Breast cancer dissemination promoted by a neuregulin-collagenase 3 signalling node.

doi: 10.1038/onc.2015.337

Figure Lengend Snippet: Figure 5. NRG controls collagenase 3 levels through the ERK1/2 route. (A) Expression of different antigens used as readouts of activation of different signalling routes. Lysates from MCF7 cells treated or not with NRG were incubated with the antibody arrays to detect active (phosphorylated) forms of different antigens which participate in relevant signalling pathways. (B) Quantitation of the array data shown in (A). (C) Effect of blockade of the PI3K, mTOR and MEK1/2 on NRG-induced collagenase 3 upregulation. MCF7 cells were preincubated for 1 h with LY294002 (10 μm), U0126 (10 μm), Rapamycin (100 nM) or Ku0063794 (1 μm), and then treated with NRG (10 nM). The levels of collagenase 3, pERK1/2, pAKT, pS6 and GAPDH were analyzed by western blotting. (D) Effect of a dominant-negative form of ERK1/2 on NRG-induced upregulation of collagenase 3. MCF7 cells transfected with the vector pLNK-ERK2K52R coding for a dominant negative form of ERK1/2 (HA-tagged ERK2K52R) were incubated with NRG and collagenase 3 expression, ERK1/2, HA-ERK2K52R and GAPDH analyzed in 50 μg of cell extracts. (E) Time course analysis of the effect of NRG on collagenase 3 production, and pERK1/2 and ERK1/2 levels. Fifty micrograms of protein from each sample were used in western blotting. (F) Quantitative analyses of the collagenase 3 and pERK1/2 data shown in (E). (G) Western blotting analyses of collagenase 3, pERK1/2 and ERK1/2 expression in patient samples. Collagenase 3 was immunoprecipitated from 2 mg of protein. Assessment of pERK1/2 and ERK1/2 was carried out directly on 50 μg of protein from each sample. (H) Quantitative regression plotting of the data obtained in panel (G).

Article Snippet: Preparation of cell lysates for immunoprecipitation or western analyses was as described.39,40 For the antibody arrays, 600 μg of MCF7 cell lysates treated or not with NRG were hybridized to ARY003B antibody arrays (R&D Systems, Abingdon, UK).

Techniques: Expressing, Activation Assay, Incubation, Quantitation Assay, Western Blot, Dominant Negative Mutation, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Oncogene

Article Title: Breast cancer dissemination promoted by a neuregulin-collagenase 3 signalling node.

doi: 10.1038/onc.2015.337

Figure Lengend Snippet: Figure 6. Transcription-dependent upregulation of collagenase-3 levels by NRG. (a) qPCR analysis of collagenase 3 mRNA levels in MCF7 cells treated for different times with NRG. A set of samples were preincubated with 10 μm of the MEK1/2 inhibitor U0126. (b) Transcriptional analysis of the collagenase 3 promoter. MCF7 cells were transfected with 1 μg of each of the reporter plasmids indicated together with 100 ng of the pCDN3-renilla plasmid (used to normalize for transfection efficiency). Luminescent data are presented as mean ± s.d. (c) RT–PCR analyses of the expression of distinct transcription factors in MCF7 cells. (d) Description of the promoter region mainly responsible for the regulation of collagenase 3 by NRG. Sequences putatively recognized by the transcription factors YY1 and SBF-1 are shown, as well as the mutant sequences used to analyze the relevance of these regions in the regulation of collagenase 3 expression. (e) Identification of the region responsible for the upregulation of collagenase 3 expression upon activation of NRG receptors. MCF7 cells were cotransfected with 1 μg of the indicated plasmids corresponding to wild-type −492+2 collagenase 3 pGL2-B or SBF-1 and YY1 mutants, together with 100 ng of pCDNA3- renilla. Data are plotted as in (b). (f) A schematic representation of the mechanism of activation of collagenase 3 by NRG.

Article Snippet: Preparation of cell lysates for immunoprecipitation or western analyses was as described.39,40 For the antibody arrays, 600 μg of MCF7 cell lysates treated or not with NRG were hybridized to ARY003B antibody arrays (R&D Systems, Abingdon, UK).

Techniques: Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis, Activation Assay