Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants reduces CTCs. MDA-MB-231 cells with constitutive sh mdm2 , sh mdmx , or mir30 shRNA-expressing vector were implanted into the mammary fat pads of 6-week-old female NSG mice. a Western blot analysis of MDM2, MDMX, and mtp53 protein levels from 50 μg of whole-cell lysates from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively) prior to mammary fat pad implantation. Actin is shown as a loading control. b Box-and-whisker plot represents the numbers of CTCs per milliliter from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells engrafted into animals. The number of CTCs was determined by flow cytometry, and the total events were counted (gates were set by the GFP signal intensity and cell size). The number of CTCs per milliliter was obtained by dividing the number of positive events by blood volume from individual animals. The adjusted p value was obtained with two-tailed, two-sample t tests using a permutation test. c Representative fluorescence-activated cell sorting plots showing GFP-positive events in different mouse groups

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Plasmid Preparation, Western Blot, Control, Whisker Assay, Flow Cytometry, Two Tailed Test, Fluorescence, FACS

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants does not significantly reduce primary tumor growth. a Primary tumor volumes of 231.mir30.vector ( n = 6), 231.sh mdm2 ( n = 7), and 231.sh mdmx (n = 7) engrafted animals were measured using calipers over 36 days. b The endpoint tumor volumes were determined on dissected masses at the time of necropsy. c mRNA levels of mdm2 and mdmx normalized to gapdh in primary tumors were determined by real-time qRT-PCR. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = nonsignificant. The p value was calculated using two-tailed unpaired t tests. d Protein expression of MDM2, MDMX, and mtp53 from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx engrafted primary tumors were determined by Western blot analysis. Three tumors per group were used, and actin is shown as a loading control

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Expressing, Western Blot, Control

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 provoke in vitro MDA-MB-231 cell migration without altering cell proliferation. a Representative Western blot demonstrating the levels of MDM2, MDMX, and mtp53 in 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively). Fifty micrograms of lysate was loaded per lane. Actin was used as a loading control. b The number of cells was determined by hemocytometer cell counting. Cells ( n = 50,000) were seeded in triplicate, and cell counting was performed at 2, 4, 5, and 6 days. Dots represent mean values, and error bars represent SD. Experiments were carried out with three biological replicates. c Wound closure was observed by phase-contrast microscopy and photographed at 0 and 12 h. One representative image from each group at 0 and 12 h is shown. d The wound area was measured by using NIS-Elements software (Nikon Instruments, Melville, NY, USA). The percentage of wound closure was quantified from four independent biological experiments. The p value was obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: In Vitro, Migration, Western Blot, Plasmid Preparation, Control, Cell Counting, Microscopy, Software, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX knockdown in primary tumors blocks the transcription of CXCR4 and PTGS2 . The 231.mir30.vector-, 231.sh mdm2 -, and 231.sh mdmx -derived primary tumors were lysed and used for total RNA extraction and complementary DNA synthesis. a Microarray analysis revealed selected tumor metastasis-related genes that were either up- or downregulated in 231.sh mdm2 and 231.sh mdmx compared with 231.mir30 vector. Fold changes were gated either > 2 or < 0.5. Two tumor samples per group were used for the analysis. b From the respective cells derived from all the primary tumors, the total CXCR4 and PTGS2 levels were determined by real-time qRT-PCR, and these were compared with those of the parental cells grown in culture. The bars represent mean values, and error bars represent SD. The p values were obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Derivative Assay, RNA Extraction, DNA Synthesis, Microarray, Quantitative RT-PCR, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDM2 knockdown in ERα + T47D orthotopic transplant reduces tumor volume. a T47D cells with inducible sh mdm2 or mir30 shRNA-expressing control vector were treated with 4 μg/ml doxycycline (Dox) for 10 days to induce and maintain shRNA expression. Western blot shows the levels of MDM2, MDMX, E-cadherin, and mtp53 with Dox treatment (lanes 1 and 2) prior to mammary fat pat implantation. Actin was used as a loading control. b Animals were provided with 2 mg/ml Dox and 8 μg/ml E 2 in their drinking water during the entire experiment. Primary tumor growth was measured over a period of 60 days using calipers *** p < 0.001 calculated by two-tailed unpaired t test. c The experimental endpoint tumor volume was determined at the time of necropsy. d mdm2 mRNA expression in primary tumors was determined by real-time qRT-PCR. The p value was determined by two-tailed unpaired t test. e E-cadherin, MDM2, MDMX, and mtp53 protein levels from primary tumors were determined by Western blot analysis. Actin was used as the loading control. f Representative H&E staining images of T47D.vector and T47D.sh mdm2 under 200× and 1000× magnification. T represents Tumor; nm represents normal mammary fat pad; M represents muscle; arrowhead depicts tumor cells infiltrating into muscle layer

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Control, Plasmid Preparation, Western Blot, Two Tailed Test, Quantitative RT-PCR, Staining

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: Comparative levels of CXCR4 in ERα + T47D and TNBC MDA-MB-231 tumors. a CXCR4 RNA expression normalized to gapdh with MDM2 knockdown was determined using real-time qRT-PCR. 231.mir30.vector ( n = 6), T47D.mir30.vector ( n = 10), and T47D.sh mdm2 ( n = 9) tumor samples were analyzed. The RNA level of CXCR4 was set as 1 for the 231.mir30.vector group, and T47D samples were expressed relative to 231.mir30.vector values. b Protein expression of CXCR4 was compared from parental cell lines and two tumors from each group. Representative Western blot demonstrating protein levels of CXCR4 and actin in MDA-MB-231 and T47D groups shown using two gels (lanes 1–9 and 10–16, with tumor 2 for 231.sh mdmx used in lane 10 as a common reference)

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: RNA Expression, Knockdown, Quantitative RT-PCR, Plasmid Preparation, Expressing, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 in TNBC promote metastasis, and in ERα + breast cancer MDM2 promotes proliferation. In TNBC, MDMX promotes expression of CXCR4 and PTGS2 with associated release of CTCs but no increase in cell proliferation. In ERα + breast cancer, estrogen stimulates MDM2 expression with no influence on CXCR4 and causes an increase in cell proliferation without correlated metastasis

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Expressing

Journal: BMC Genomics

Article Title: Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

doi: 10.1186/1471-2164-13-445

Figure Lengend Snippet: Genetic map of the sigH-rshA operon, its Northern hybridization analysis in C. glutamicum RES167 and its deletion derivatives. A . Genetic map of the sigH-rshA region showing locations and sizes of deletions in the chromosomes of strains C. glutamicum Δ rshA , DN2 and AS1, predicted sizes of respective sigH-rshA and rshA transcripts (arrows) and locations of probes used for Northern hybridizations. Promoters are indicated with bent arrows and the terminator with a hairpin symbol. B . Northern blot using a sigH probe (left panel) and an rshA probe (right panel) hybridized with total RNA extracted from: RES167 cells (lane 1); DN2 cells (Δ sigH deletion; lane 2); AS1 cells (Δ sigHrshA deletion; lane 3). The estimated lenghts of the detected transcripts (left) and their designations (right) are indicated. The sizes of the fragments in the RNA marker are indicated with arrows.

Article Snippet: The blotting was performed with total RNA prepared from C. glutamicum RES167 (restriction-deficient variant derived from the ATCC 13032 type strain and its derived deletion mutant strains DN2 (carrying a deletion within sigH ) and AS1 (carrying a complete deletion of sigHrshA ).

Techniques: Northern Blot, Hybridization, Marker

Journal: BMC Genomics

Article Title: Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

doi: 10.1186/1471-2164-13-445

Figure Lengend Snippet: Microarray analysis of the C. glutamicum RES167 strain compared with its Δ rshA mutant DN2. Ratio/intensity plot obtained from the DNA microarray comparing the transcriptomes of RES167 and DN2 is shown. Total RNA was isolated from two biological replicates grown in minimal CGXII medium to the exponential phase and used for hybridization. Genes with increased amounts of mRNA in the Δ rshA strain have positive ratios, while genes with a higher mRNA amount in the RES167 strain have negative ratios, indicated with green diamonds (upregulated) or red triangles (downregulated) respectively; those not exhibiting differential expression are indicated with grey spots. M values of higher than +0.6 or lower than −0.6 (corresponding to fold changes of 1.52 and 0.66, respectively) were considered to be significant. The relevant genes are indicated by their names or desigations from the C. glutamicum genome sequence (GenBank NC_006958), underlined genes were previously described as SigH-dependent.

Article Snippet: The blotting was performed with total RNA prepared from C. glutamicum RES167 (restriction-deficient variant derived from the ATCC 13032 type strain and its derived deletion mutant strains DN2 (carrying a deletion within sigH ) and AS1 (carrying a complete deletion of sigHrshA ).

Techniques: Microarray, Mutagenesis, Isolation, Hybridization, Quantitative Proteomics, Sequencing

Journal: BMC Genomics

Article Title: Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

doi: 10.1186/1471-2164-13-445

Figure Lengend Snippet: Genes with enhanced expression in C. glutamicum Δ rshA compared with C . glutamicum RES167 (reference) sorted by function

Article Snippet: The blotting was performed with total RNA prepared from C. glutamicum RES167 (restriction-deficient variant derived from the ATCC 13032 type strain and its derived deletion mutant strains DN2 (carrying a deletion within sigH ) and AS1 (carrying a complete deletion of sigHrshA ).

Techniques: Expressing, Sequencing, Methylation, Ubiquitin Proteomics, Membrane, Luciferase

Journal: BMC Genomics

Article Title: Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

doi: 10.1186/1471-2164-13-445

Figure Lengend Snippet: Relative transcript levels of selected potential SigH-dependent genes in C. glutamicum Δ rshA/ C. glutamicum RES167 measured by q-RT-PCR. The data obtained for the RES167 strain served as a reference and the respective values were set to 1.0 on the logarithmic scale. Three biological replicates for the Δ rshA strain and four replicates for the RES167 strain were analysed in duplicate. SD values are shown as error bars.

Article Snippet: The blotting was performed with total RNA prepared from C. glutamicum RES167 (restriction-deficient variant derived from the ATCC 13032 type strain and its derived deletion mutant strains DN2 (carrying a deletion within sigH ) and AS1 (carrying a complete deletion of sigHrshA ).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

doi: 10.1186/1471-2164-13-445

Figure Lengend Snippet: Plasmids and bacteria used in this work

Article Snippet: The blotting was performed with total RNA prepared from C. glutamicum RES167 (restriction-deficient variant derived from the ATCC 13032 type strain and its derived deletion mutant strains DN2 (carrying a deletion within sigH ) and AS1 (carrying a complete deletion of sigHrshA ).

Techniques: Bacteria, Plasmid Preparation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Journal of Biological Chemistry

Article Title: Integrin α2-mediated ERK and Calpain Activation Play a Critical Role in Cell Adhesion and Motility via Focal Adhesion Kinase Signaling

doi: 10.1074/jbc.m600787200

Figure Lengend Snippet: FIGURE 2. Adhesion of GEO cells to CN IV induces tyrosine phosphorylation of proteins (A), FAK cleavage (B), FAK dephosphorylation by P1E6 (C), and ERK activation (D). GEO cells were trypsinized, incubated with soy bean trypsin inhibitor for 1 h at 37 °C, and either kept in suspension or replated to dishes coated with CN IV (5 g/ml) in SM medium. Cells were allowed to attach to CN IV for 20 or 60 min at 37 °C. Cellular lysates from cells in suspension (S) and cells attached (A) were analyzed for phosphotyrosine proteins by immunoblotting with antiphosphotyrosine antibody 4G10 (A). The immunoblots were analyzed for activation of FAK (B; upper left) or for FAK protein by using rabbit anti-human N-terminal FAK antibody (B; lower left). The right panel shows that both 125-kDa FAK and its cleaved 90-kDa form are immunoprecipitated and immunoblotted by N-terminal FAK antibody. GEO cells in SM medium were incubated either with mouse IgG1 or with integrin 2-blocking mAb (clone P1E6) at dilution 1:50 for 30 min at 37 °C. Subsequently, cells were transferred to dishes precoated with CN IV and incubated for 1 h at 37 °C. The cell lysates were analyzed by immunoblotting for pFAK and FAK using specific antibodies (C). Cellular lysates from cells in suspension (S) and cells attached (A) were also analyzed for ERK (Tyr204) by immunoblotting with specific antibody (D). Molecular weight standards are indicated in A. The positions of pFAK (125 kDa) and its major cleaved fragment (90 kDa), ERK1 (44 kDa), and ERK2 (42 kDa) are indicated by the arrowheads. Total FAK and ERK panels are shown for equal loading.

Article Snippet: The polyclonal anti-FAK phosphospecific antibodies were provided by BIOSOURCE (Camarillo, CA), whereas the rabbit anti-human C-terminal FAK (SC-558), anti-humanN-terminal FAK (SC-557), ERK, phosphorylated ERK1/2, and cyclin E antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Incubation, Suspension, Western Blot, Immunoprecipitation, Blocking Assay, Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Integrin α2-mediated ERK and Calpain Activation Play a Critical Role in Cell Adhesion and Motility via Focal Adhesion Kinase Signaling

doi: 10.1074/jbc.m600787200

Figure Lengend Snippet: FIGURE 8. Effect of U0126, cyto D, and dominant negative mutants on ERK activation. GEO cells were treated either with Me2SO or with MEK inhibitor, U0126, for 24 h in SM medium. After trypsinization, cells were seeded on CN IV-coated microarray wells. Cells were allowed to attach for 3 h, and then micromotion was recorded by the ECIS technique. Results show the inhibitory effect of U0126 on micromotion (right) as compared with control (left) (A). B shows the inhibitory effect of U0126 on ERK phosphorylation as determined by Western blot analysis using either phosphospecific antibody (Y204; top) or ERK antibody (bottom). In C, GEO cells were treated either with Me2SO (control) or with cyto D for 24 h. Aftertrypsinization,cellsweretransferredtoCNIV-precoateddishesandincubatedat37 °CinthepresenceofMe2SOorcytoDfor1h.LysateswerecollectedandanalyzedbyWestern blot using an antibody specific to phospho-ERK or total ERK, as indicated. In D–F, GEO cells were transfected either with empty vector or with the dominant negative FRNK, Y861F, or -calpain mutant for 48 h, respectively. The cell lysates were analyzed by using specific antibodies for phospho-ERK (top) or ERK (bottom).

Article Snippet: The polyclonal anti-FAK phosphospecific antibodies were provided by BIOSOURCE (Camarillo, CA), whereas the rabbit anti-human C-terminal FAK (SC-558), anti-humanN-terminal FAK (SC-557), ERK, phosphorylated ERK1/2, and cyclin E antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Dominant Negative Mutation, Activation Assay, Microarray, Control, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: miRNAs differentially expressed between A375R and A375 cells and KEGG pathway analysis of putative targets. a Heatmap showing miRNA up-regulated (red = expression above the mean) and down-regulated (blue = expression below the mean) in dabrafenib-resistant A375R cells as compared with dabrafenib-sensitive A375 cells (SAM analysis; FC ≥ 2, FDR = 0%). miRNAs are indicated according to annotation provided by Affymetrix. b Putative target genes of differentially expressed miRNAs were obtained from TargetScan and used for KEGG pathway enrichment analysis. Only pathways with an adjusted P value <0.01 were considered and listed according to a decreasing value of the combined score. c, d Expression of miRNAs down-regulated (c) or up-regulated (d) in A375R cells according to microarray results was validated using specific TaqMan® MicroRNA Assays. The data were normalized to the level of RNU44 in each sample and expressed as 2 -ΔCt x10 5 values. Each value represents the arithmetic mean of at least three independent experiments performed with duplicate samples. Bars, standard error of the mean (SEM). ** P <0.01 and * P <0.05, A375R versus ( vs ) A375.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Expressing, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: miR-126-3p is up-regulated by dabrafenib only in drug-sensitive cells and inhibits their proliferation. a, b Melanoma cells were incubated with 100 nM dabrafenib (DAB) or with DMSO alone and after 48 h of culture miR-126-3p expression was evaluated by qRT-PCR. 2 -ΔCt x10 5 values calculated relative to RNU44 as the internal reference are shown. Each value represents the arithmetic mean of three independent experiments performed with duplicate samples. Bars, SEM. ** P <0.01 and * P <0.05, DAB vs matched DMSO; ## P <0.01 and # P <0.05, resistant cells/DMSO vs parental cells/DMSO. c A375 and SK-Mel28 were transiently transfected with 50 nM pre-miR-126-3p or pre-miR-CTRL, cultured for six days and then assayed for proliferation by the MTT assay. Data are expressed in terms of absorbance at 595 nM. Each value represents the arithmetic mean of three (A375) or four (SK-Mel28) independent experiments performed with triplicate samples. Bars, SEM. * P <0.05, pre-miR-126-3p vs pre-miR-CTRL.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Transfection, Cell Culture, MTT Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: ADAM9 silencing delays the development of resistance to dabrafenib. A375 cells were seeded into 96-well plates and every eight days transfected with 50 nM siADAM9 or siCTRL and treated with 100 nM dabrafenib (DAB) or DMSO. Cell cultures were photographed and processed for quantitative analysis of proliferation on day 0 (i.e after the first transfection), 8, 16 and 24. Images from a representative experiment are shown. b Quantitative analysis of proliferation of cell cultures described in (a). Crystal violet was solubilized and absorbance was read at 595 nm. Each value represents the arithmetic mean of three independent experiments performed with triplicate cultures. Bars, SEM. ** P <0.01, siADAM9 vs matched siCTRL; §§ P <0.01, siCTRL/DAB/Day 8 vs siCTRL/DMSO/Day 8; †† P <0.01, siADAM9/DAB/Day 8 vs siADAM9/DMSO/Day8; ## P <0.01, siCTRL/DAB/Day 16 vs siCTRL/DAB/Day 8; ⁋⁋ P <0.01, siCTRL/DAB/Day 24 vs siCTRL/DAB/Day 16.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Transfection

Journal: Scientific Reports

Article Title: Single ingestion of soy β-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects

doi: 10.1038/srep28183

Figure Lengend Snippet: Five-week old male mice were acclimated to the casein high-fat diet for 3 days. After 24 h fasting, mice were fed with either the casein or β-conglycinin (β-con) high-fat diet for 6 h as shown in . Using liver total RNA, microarray analysis was performed according to the manufacturer’s instructions (n = 4). ( a ) GO terms associated with the genes that were up-regulated in the β-conglycinin group. FDR-corrected P values were defined by the modified Fisher’s exact test with the Benjamini and Hochberg FDR correction. FDR-corrected P values < 0.05 are shaded in gray. ( b ) GO terms associated with the genes that were down-regulated in the β-conglycinin group. ( c ) Heatmap shows top twenty genes up-regulated or down-regulated by β-conglycinin ingestion from DNA microarray experiments. Green color indicates upregulation and red indicates downregulation in response to ingestion of two types of diet. ( d ) RT-qPCR was performed using total RNA used in DNA microarray experiments (n = 5). Relative expression levels of FGF21 and ATF4 mRNA in the liver are normalized to cyclophilin mRNA levels and are shown as fold induction to expression levels in the casein group. Serum FGF21 concentrations are determined by ELISA. All data are expressed as means ± SD (n = 5). ** P < 0.01 determined by two-tailed Student’s t -test.

Article Snippet: Cross-linking reactions were stopped by adding glycine to 0.125 M. Liver nuclei were isolated with a Dounce homogenizer in hypotonic solution followed by centrifugation at 4000 × g for 1 min. Chromatin immunoprecipitation assays using liver nuclei were performed using a ChIP assay kit (Upstate Biotechnology) and anti-ATF4 antibody (5 μg, Santa Cruz Biotechnology, Inc.) or control rabbit IgG (Millipore Corporation) or anti-acetyl-Histone H3 (Lys9 [Upstate Biotechnology]).

Techniques: Microarray, Modification, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Single ingestion of soy β-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects

doi: 10.1038/srep28183

Figure Lengend Snippet: Five-week-old male WT and FGF21 KO mice were acclimated to the casein high-fat diet for 7 days, and then fed with either the casein or β-conglycinin (β-con) diet for 9 weeks. After 6 h of starvation, mice were sacrificed. ( a ) Body weight of mice. All data are expressed as means ± SD (n = 6–8). ( b ) Total food consumption over 9 weeks. ** P < 0.01 determined by two-tailed Student’s t -test. ( c ) Body weight gain over 9 weeks. ( d ) Adipose tissue (epididymal and subcutaneous white adipose tissue, and brown adipose tissue) weights of mice. ( e ) Expression levels of FGF21 mRNA in the liver are normalized to cyclophilin mRNA levels and are shown as fold induction to expression levels in the casein group of WT mice. * P < 0.05 determined by two-tailed Student’s t -test. ( f‒k ) Serum FGF21, glucose, insulin, triglyceride (TG), total cholesterol, and Igf-1 concentrations. ( l ) Liver TG contents. ( m ) Liver total cholesterol contents. ( n ) Expression levels of ATF4 target gene mRNA in the liver are normalized to cyclophilin mRNA levels and are shown as fold induction to expression levels in the casein group of WT mice. ( o ) Expression levels of mRNA related to lipid metabolism in the liver are normalized to cyclophilin mRNA levels and are shown as fold induction to expression levels in the casein group of WT mice. ( p ) Expression levels of mRNA related to bile acid metabolism in the ileum are normalized to β-actin mRNA levels and are shown as fold induction to expression levels in the casein group of WT mice. ( q‒s ) Expression levels of mRNA related to lipolysis in adipose tissues are normalized to 18S ribosomal RNA levels and are shown as fold induction to expression levels in the casein group of WT mice.

Article Snippet: Cross-linking reactions were stopped by adding glycine to 0.125 M. Liver nuclei were isolated with a Dounce homogenizer in hypotonic solution followed by centrifugation at 4000 × g for 1 min. Chromatin immunoprecipitation assays using liver nuclei were performed using a ChIP assay kit (Upstate Biotechnology) and anti-ATF4 antibody (5 μg, Santa Cruz Biotechnology, Inc.) or control rabbit IgG (Millipore Corporation) or anti-acetyl-Histone H3 (Lys9 [Upstate Biotechnology]).

Techniques: Two Tailed Test, Expressing

Journal: Scientific Reports

Article Title: Single ingestion of soy β-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects

doi: 10.1038/srep28183

Figure Lengend Snippet: ( a ) Mouse primary hepatocytes were transfected with 200 ng of one of reporter constructs, either pCMV 3xFLAG (mock) or pCMV 3xFLAG mATF4, and a β-galactosidase construct. After incubation for 48 h, luciferase and β-galactosidase activities were determined. Normalized luciferase values were determined by dividing the luciferase activity by the β-galactosidase activity. Relative activities are shown as fold induction to expression levels in the mFGF21-1133 bp together with pCMV 3xFLAG. ( b ) HEK293T cells were transfected as described in . In the fourth group the cells were transfected with 350 ng of pCMV 3xFLAG mATF4 and 515 ng of pCMV 3xFLAG mATF4DN (a dominant negative form of mATF4). The structure of mATF4 and mATF4DN are depicted. Relative luciferase activities are shown as fold induction to expression levels in the mFGF21-1133 bp together with pCMV 3xFLAG. One-way analysis of variance followed by the Bonferroni procedure was used. Different superscript letters denote statistical significance ( P < 0.05). ( c , d ) Eight-week-old mice were injected through the tail vein with 1 × 10 9 pfu of adenovirus bearing either Lac Z or mATF4DN. After 24 h, these mice were fasted for 24 h and then re-fed the β-con diet for 6 h. Hepatic FGF21 mRNA levels are normalized to 18S rRNA levels and are shown as fold induction to expression levels in the 24 h-fasted Lac Z group. Serum FGF21 concentrations are also shown. ** P < 0.01 determined by two-tailed Student’s t -test. ( e , f ) Livers obtained from mice fed for 3 h were processed for ChIP analyses. After immunoprecipitation with anti-ATF4, anti-acetyl-histone H3, or control IgG, real-time PCR analysis was performed with a primer set covering the region (−6533 to −6470) of the promoter in the mouse FGF21 gene, or the region covering the AARE sequence (as illustrated to the right side of ). The results were represented by fold enrichment relative to control IgG. The same results were obtained in two separate experiments. * P < 0.05 by two-tailed Student’s t -test. All data are expressed as means ± SD (n = 3).

Article Snippet: Cross-linking reactions were stopped by adding glycine to 0.125 M. Liver nuclei were isolated with a Dounce homogenizer in hypotonic solution followed by centrifugation at 4000 × g for 1 min. Chromatin immunoprecipitation assays using liver nuclei were performed using a ChIP assay kit (Upstate Biotechnology) and anti-ATF4 antibody (5 μg, Santa Cruz Biotechnology, Inc.) or control rabbit IgG (Millipore Corporation) or anti-acetyl-Histone H3 (Lys9 [Upstate Biotechnology]).

Techniques: Transfection, Construct, Incubation, Luciferase, Activity Assay, Expressing, Dominant Negative Mutation, Injection, Two Tailed Test, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, Sequencing

Journal: Scientific Reports

Article Title: Single ingestion of soy β-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects

doi: 10.1038/srep28183

Figure Lengend Snippet: Five-week old mice were fasted for 24 h and then fed with the indicated high-fat diet. Using liver total RNA, RT-qPCR was performed and relative mRNA levels are normalized to cyclophilin mRNA levels. ( a ) Mice were fed with the diet for 6 h. Relative mRNA expression levels are shown as fold induction to expression levels of the indicated genes in the casein group. All data are expressed as means ± SD (n = 6). One-way analysis of variance followed by the Bonferroni procedure was used. Different superscript letters denote statistical significance ( P < 0.05). ( b ) Mice fed with 1.2 g the diet and then sacrificed at the indicated time. Relative mRNA expression levels are shown as fold induction to expression levels at 0 (24-h fasted). All data ( b–i ) are expressed as means ± SD (n = 3). * P < 0.05 and ** P < 0.01 determined by two-tailed Student’s t -test. ( c ) Portal blood serum was obtained at 1 h after feeding of the diet. ( d , e ) Mice were fed for 6 h. Relative mRNA expression levels are shown as fold induction to expression levels in the casein group. Serum FGF21 concentrations are also shown. Different superscript letters denote statistical significance ( P < 0.05). ( f , g ) Mice were fed for 6 h. Relative mRNA expression levels are shown as fold induction to expression levels in the casein group. Serum FGF21 concentrations are also shown. Different superscript letters denote statistical significance ( P < 0.05). ( h ) Mice were fed with 1.2 g of the diet and then portal blood was collected at the indicated time. Serum methionine concentrations are shown. Different superscript letters denote statistical significance ( P < 0.05). ( i , j ) ChIP analyses using anti-ATF4 or control IgG were performed as described in . The results were represented by fold enrichment relative to control IgG in 4 different groups. The same results were obtained in two separate experiments.

Article Snippet: Cross-linking reactions were stopped by adding glycine to 0.125 M. Liver nuclei were isolated with a Dounce homogenizer in hypotonic solution followed by centrifugation at 4000 × g for 1 min. Chromatin immunoprecipitation assays using liver nuclei were performed using a ChIP assay kit (Upstate Biotechnology) and anti-ATF4 antibody (5 μg, Santa Cruz Biotechnology, Inc.) or control rabbit IgG (Millipore Corporation) or anti-acetyl-Histone H3 (Lys9 [Upstate Biotechnology]).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Control

Journal: Nature Communications

Article Title: Myeloid-derived interleukin-1β drives oncogenic KRAS -NF-κΒ addiction in malignant pleural effusion

doi: 10.1038/s41467-018-03051-z

Figure Lengend Snippet: Kras -mutant tumor cells exhibit non-canonical endogenous NF-κΒ activity. Five different C57BL/6 mouse tumor cell lines with ( Kras MUT : LLC, MC38, AE17) or without ( Kras WT : B16F10, PANO2) Kras mutations were assessed for activation and inhibition of resting NF-κΒ activity in vitro. a Map of NF-κΒ reporter plasmid (NF-κΒ.GFP.Luc; p NGL ). Partial p NGL sequence at origin (1) showing κΒ-binding motifs (red) and GFP sequence (green). b Representative image and data summary ( n = 3) of area under curve of cumulative bioluminescence emitted by cells transiently transfected with reporter plasmids p CAG.LUC or p NGL . c Data summary ( n = 8) of bioluminescence emitted by PANO2 cells stably expressing p NGL reporter plasmid at 48 h after transient transfection with p C or p Kras G12C . d Data summary ( n = 5) of DNA NF-κB motif binding activity of nuclear extracts by NF-κB ELISA relative to nuclear extracts of Raji leukemia cells. e Immunofluorescent detection of Rel A and Rel B in cells grown on glass slides ( n = 3) showing increased nuclear localization of Rel B in Kras MUT cells and of Rel A in Kras WT cells (arrows). f Immunoblots of cytoplasmic and nuclear extracts for NF-κB pathway members and β-actin (representative of n = 3 independent experiments). Data presented as mean ± s.d. P , overall probability by one-way ( b ) and two-way ( d ) ANOVA or Student’s t -test ( c ). * P < 0.05 and *** P < 0.001 for the indicated comparisons by Bonferroni post-tests. g Data summary ( n = 3) of p NGL reporter activity after 4-h treatment and of cell proliferation by MTT assay after 72-h treatment in response to bortezomib, IMD-0354, or 17-DMAG. Data presented as mean ± s.d. from n = 3 replicates/data point. P , probability of no difference between cell lines by extra sum-of-squares F test. h , i Data summary of 50% inhibitory concentrations (IC 50 ) of NF-κΒ activity (by p NGL reporter activity) and cell proliferation (by MTT; g ). Data presented as mean ± s.d. from n = 3 independent experiments. P , probability of no difference by two-way ANOVA. ns and triple asterisks (***): P > 0.05 and P < 0.001, respectively, for the indicated comparisons by Bonferroni post-tests. nd not determined

Article Snippet: D-Luciferin was from Gold Biotechnology (St. Louis, MO); lentiviral shRNA and puromycin from Santa Cruz (Dallas, TX); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33528 from Sigma-Aldrich (St. Louis, MO); mouse gene ST2.0 microarrays and relevant reagents from Affymetrix (Santa Clara, CA); recombinant cytokines and growth factors from Immunotools (Friesoythe, Germany); NF-κB-binding ELISA from Active Motif (La Hulpe, Belgium); bortezomib, IMD-0354, 17-DMAG, and deltarasin from Selleckchem (Houston, TX); G418 from Applichem (Darmstadt, Germany); IL-1β and CXCL1 ELISA from Peprotech (London, UK); and primers from VBC Biotech (Vienna, Austria).

Techniques: Mutagenesis, Activity Assay, Activation Assay, Inhibition, In Vitro, Plasmid Preparation, Sequencing, Binding Assay, Transfection, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, MTT Assay

Journal: Nature Communications

Article Title: Myeloid-derived interleukin-1β drives oncogenic KRAS -NF-κΒ addiction in malignant pleural effusion

doi: 10.1038/s41467-018-03051-z

Figure Lengend Snippet: Kras -mutant tumor cells possess IL-1β-inducible NF-κΒ activity. Five different C57BL/6 mouse tumor cell lines with (LLC, MC38, AE17) or without (B16F10, PANO2) Kras mutations were assessed for inducible NF-κΒ activation in response to exogenous stimuli and for the expression of relevant receptors in vitro. a , b Representative bioluminescent images ( a ; shown are n = 3 replicates/data-point) and data summary ( b ; mean ± s.d. of n = 3 independent experiments) of cells stably expressing p NGL and pretreated with saline or 1 μM bortezomib at different time points after addition of 1 nM of the indicated NF-κΒ ligands (arrows in a and legend in b ). Note NF-κB inducibility by IL-1β and bortezomib exclusively in Kras MUT cells. ns and *** P > 0.05 and P < 0.001, respectively, for comparison between ligands indicated by colored arrows and PBS at 4 and 8 h on treatment by two-way ANOVA with Bonferroni post-tests. c , d Tnfrsf1a , Tnfrsf1b , Il1r1 , Il1a , and Il1b mRNA expression relative to Gusb by microarray ( c ) and qPCR ( d ). Shown are mean ( c ) and mean ± s.d. ( d ) of n = 5 independent technical replicates of one biologic sample. P , probability of no difference between cell lines by two-way ( c ) or one-way ( d ) ANOVA. ns, single, double, and triple asterisks (*, **, and ***): P > 0.05, P < 0.05, P < 0.01, and P < 0.001, respectively, for comparison with B16F10 cells ( c ) by Bonferroni post-tests

Article Snippet: D-Luciferin was from Gold Biotechnology (St. Louis, MO); lentiviral shRNA and puromycin from Santa Cruz (Dallas, TX); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33528 from Sigma-Aldrich (St. Louis, MO); mouse gene ST2.0 microarrays and relevant reagents from Affymetrix (Santa Clara, CA); recombinant cytokines and growth factors from Immunotools (Friesoythe, Germany); NF-κB-binding ELISA from Active Motif (La Hulpe, Belgium); bortezomib, IMD-0354, 17-DMAG, and deltarasin from Selleckchem (Houston, TX); G418 from Applichem (Darmstadt, Germany); IL-1β and CXCL1 ELISA from Peprotech (London, UK); and primers from VBC Biotech (Vienna, Austria).

Techniques: Mutagenesis, Activity Assay, Activation Assay, Expressing, In Vitro, Stable Transfection, Saline, Comparison, Microarray

Journal: Nature Communications

Article Title: Myeloid-derived interleukin-1β drives oncogenic KRAS -NF-κΒ addiction in malignant pleural effusion

doi: 10.1038/s41467-018-03051-z

Figure Lengend Snippet: Mutant Kras drives basal and IL-1β-induced non-canonical NF-κB signaling and drug resistance. a Rel A and Rel B binding of nuclear extracts of MC38 cells stably expressing a control plasmid (p C ), a mutant dominant-negative form of ΙκΒα (p ΙκΒα DN), control shRNA (sh C ), or anti- Kras shRNA (sh Kras ) relative to Raji leukemia cells by NF-κB ELISA ( n = 3 experiments). b Immunofluorescent detection of Rel A and Rel B in MC38 cells showing increased nuclear Rel B (arrows) and its disappearance in cells expressing sh Kras . c IKKα immunoblots of cytoplasmic and nuclear extracts of LLC and MC38 cells expressing sh C or sh Kras . ( n = 3 experiments). d , e MTT data ( n = 3 replicates/data-point) and mean (95% CI) IC 50 values ( n = 3 experiments) of MC38 cells stably expressing sh C or sh Kras treated with bortezomib ( d ) or IMD-0354 ( e ) for 72 h. P , probability of no difference between cell lines by extra sum-of-squares F test. f , g Bioluminescent detection of NF-κΒ activity in Kras MUT ( f ) and Kras WT ( g ) cells stably expressing p NGL and the indicated vectors during 4-h incubation with PBS or 1 nM TNF or IL-1β ( n = 3 experiments). h , i Data summary ( h ; n = 6 mice/group) and images ( i ) of C57BL/6 mice at 0 and 4 h after intrapleural injection of MC38 or PANO2 cells stably expressing pNGL and the indicated vectors. j Il1r1 mRNA expression by qPCR of Kras MUT and Kras WT cells stably expressing the indicated vectors. k Immunoblots of protein extracts of MC38 and PANO2 cells stably expressing the indicated vectors for NF-κB members after 4-h incubation with PBS or 1 nM TNF or IL-1β ( n = 3 experiments). l The above extracts were subjected to EMSA. Super-shift EMSA was performed with the indicated antibodies. IgG antibody served as negative control. Data are presented as mean ± s.d. P , probability of no difference between cell lines by two-way ANOVA. Single, double, and triple asterisks (*, **, and ***): P < 0.05, P < 0.01, and P < 0.001, respectively, for comparison with p C or sh C ( a , f , h , j ) or with PBS ( g ) by Bonferroni post-tests

Article Snippet: D-Luciferin was from Gold Biotechnology (St. Louis, MO); lentiviral shRNA and puromycin from Santa Cruz (Dallas, TX); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33528 from Sigma-Aldrich (St. Louis, MO); mouse gene ST2.0 microarrays and relevant reagents from Affymetrix (Santa Clara, CA); recombinant cytokines and growth factors from Immunotools (Friesoythe, Germany); NF-κB-binding ELISA from Active Motif (La Hulpe, Belgium); bortezomib, IMD-0354, 17-DMAG, and deltarasin from Selleckchem (Houston, TX); G418 from Applichem (Darmstadt, Germany); IL-1β and CXCL1 ELISA from Peprotech (London, UK); and primers from VBC Biotech (Vienna, Austria).

Techniques: Mutagenesis, Binding Assay, Stable Transfection, Expressing, Control, Plasmid Preparation, Dominant Negative Mutation, shRNA, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Incubation, Injection, Negative Control, Comparison

Journal:

Article Title: Up-Regulation of Activating Transcription Factor-5 Suppresses SAP Expression to Activate T Cells in Hemophagocytic Syndrome Associated with Epstein-Barr Virus Infection and Immune Disorders

doi: 10.2353/ajpath.2008.080440

Figure Lengend Snippet: ATF5, not ATF3/7, plays an important role in LMP1-mediated suppression of SAP. A: The H9 cell line stably transfected with LMP1 and with control vector (pSG5) was harvested to perform cDNA microarray analysis. The results shown were the ratio of transcriptional repressors of LMP1-expressed H9 to pSG5-H9 cells. The criteria for up-regulation and down-regulation were a ratio of up to 2-fold and less than 0.5-fold, respectively. B: To confirm and identify the candidate repressor, the shRNA (pSUPER-EGFP) of ATF3, ATF5, and ATF7 were separately transfected to LMP1-expressed H9 cells. The Western blotting showed the expression of SAP only in ATF5 shRNA-transfected H9 T cells. The expression of ATF3, ATF5, and ATF7 revealed the specificity of each shRNA. The expression of actin was shown in the bottom to ensure equal protein loading. C: Luciferase reporter assay for the SAP promoter. Equal numbers of Jurkat T cells with high expressing level of SAP were used and transiently transfected with pSG5-LMP1, reporter construct, and shRNA. The fold induction was relative to Jurkat control cells transfected with reporter vector (pGL3-Basic).

Article Snippet: Anti-LMP1 mouse monoclonal antibody (CS1-4; DAKO, Glostrup, Denmark), anti-ATF5 antibody (Imgenex), anti-SAP rabbit antibody, anti-α-actin goat antibody (Santa Cruz Biotechnology), and NF-κB inhibitor Bay11-7082 (Sigma) were used.

Techniques: Stable Transfection, Transfection, Control, Plasmid Preparation, Microarray, shRNA, Western Blot, Expressing, Luciferase, Reporter Assay, Construct

Journal:

Article Title: Up-Regulation of Activating Transcription Factor-5 Suppresses SAP Expression to Activate T Cells in Hemophagocytic Syndrome Associated with Epstein-Barr Virus Infection and Immune Disorders

doi: 10.2353/ajpath.2008.080440

Figure Lengend Snippet: Overexpression of ATF5 induced secretion of Th1 cytokines via TRAF/NF-κB pathway. A: Jurkat and H9 T cell lines were transiently transfected with ATF5 or LMP1 plasmids. After 48 hours, cells were harvested and protein lysates were collected. Overexpression of ATF5 and suppression of SAP were observed by Western blotting assay. B: Jurkat and H9 cells (5 × 105 cells/5 ml of medium) were transiently transfected with ATF5 and LMP1 plasmids. After 48 hours, supernatants were collected to perform ELISA assay of TNF-α and IFN-γ. The results shown are representative of three independent experiments. C: Jurkat cells were co-transfected with reconstituted plasmid, SAP promoter-driven luciferase reporter construct, and control plasmid, and performed luciferase reporter assay. The fold induction was relative to control reporter plasmid (pGL3-Basic). D: The TRAF2/5 dominant-negative plasmid (TRAF2DN/5DN) could express mutated protein and effectively block the effect of LMP1 signaling on the up-regulation of ATF5 and suppression of SAP. E: LMP1-expressed H9 cells were treated with indicated dose Bay 11-7082 for 24 hours, and expression levels ATF5, SAP, and LMP1 were examined by Western blotting assay. Actin level was used for loading control. F: LMP1-expressed H9 cells were transfected with IκB dominant-negative plasmid (IκBDN) for 24 hours to inhibit NF-κB activation. The expression levels of ATF5, SAP, and LMP1 detected by Western blotting assay were also showed here.

Article Snippet: Anti-LMP1 mouse monoclonal antibody (CS1-4; DAKO, Glostrup, Denmark), anti-ATF5 antibody (Imgenex), anti-SAP rabbit antibody, anti-α-actin goat antibody (Santa Cruz Biotechnology), and NF-κB inhibitor Bay11-7082 (Sigma) were used.

Techniques: Over Expression, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Construct, Control, Reporter Assay, Dominant Negative Mutation, Blocking Assay, Expressing, Activation Assay

Journal:

Article Title: Up-Regulation of Activating Transcription Factor-5 Suppresses SAP Expression to Activate T Cells in Hemophagocytic Syndrome Associated with Epstein-Barr Virus Infection and Immune Disorders

doi: 10.2353/ajpath.2008.080440

Figure Lengend Snippet: Activated primary T cells or engaged T-cell lines showed up-regulation of ATF5. A: Primary T cells were isolated from whole blood by anti-CD3 beads. A half of primary T cells were treated with 2 μmol/L PHA to stimulate and activate T cells for 18 hours. LMP1 was transfected into naïve or PHA-stimulated primary T cells. After another 24 hours, protein lysates were collected for Western blotting, and supernatants were subjected to ELISA assay for IFN-γ and TNF-α. ATF5 up-regulation and SAP down-regulation in PHA-stimulated and LMP1-expressed T cells were shown by Western blotting analysis. B: The IFN-γ and TNF-α were dramatically induced by PHA and LMP1. C and D: The primary T cells were treated with or without 2 μmol/L PHA for different time courses. The cell lysates and supernatants were collected to perform Western blotting and ELISA assay. The ATF5 was enhanced immediately, but decreased after treating PHA for 2 hours. The SAP expression was suppressed by PHA and restored at 48 hours. After the ATF5 up-regulation and SAP down-regulation, the IFN-γ and TNF-α secretion were induced. E: Jurkat and H9 cell lines were co-treated with anti-CD3 and anti-CD28 antibodies for 6 hours to activate T cells by engaging T-cell receptor. The induction of ATF5 and inhibition of SAP were observed in engaged T-cell lines and LMP1-expressed cells (left). While pretreating with ATF5-shRNA for 72 hours, the CD3/CD28-induced ATF5 up-regulation and SAP suppression were reversed in Jurkat T cells (right).

Article Snippet: Anti-LMP1 mouse monoclonal antibody (CS1-4; DAKO, Glostrup, Denmark), anti-ATF5 antibody (Imgenex), anti-SAP rabbit antibody, anti-α-actin goat antibody (Santa Cruz Biotechnology), and NF-κB inhibitor Bay11-7082 (Sigma) were used.

Techniques: Isolation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Inhibition, shRNA

Journal:

Article Title: Up-Regulation of Activating Transcription Factor-5 Suppresses SAP Expression to Activate T Cells in Hemophagocytic Syndrome Associated with Epstein-Barr Virus Infection and Immune Disorders

doi: 10.2353/ajpath.2008.080440

Figure Lengend Snippet: The region −420 to −210 of SAP promoter was responsible for inhibition by ATF5. A: A series of promoter regions −1250 to +11, −420 to +11 (a+b), −210 to +11 (b), and −420 to −210 (a) of SAP promoter were constructed to perform reporter assay. B: Luciferase reporter assay of the SAP promoter were analyzed for the LMP1-repressable region. Data were relative to control cells and expressed as the mean + SD of quadruplicate sample. C: ChIP assay demonstrated binding of ATF5 to the promoter region of SAP. Control H9 and LMP1-expressed H9 cells were collected and ChIP assay was performed with anti-ATF5 and normal serum (negative control for precipitation). Precipitated DNA fragments were amplified by PCR using specific primers for the region −420 to −210 and region −210 to +11 of SAP promoter. Input material corresponds to 1% of the DNA material subjected to ChIP assay.

Article Snippet: Anti-LMP1 mouse monoclonal antibody (CS1-4; DAKO, Glostrup, Denmark), anti-ATF5 antibody (Imgenex), anti-SAP rabbit antibody, anti-α-actin goat antibody (Santa Cruz Biotechnology), and NF-κB inhibitor Bay11-7082 (Sigma) were used.

Techniques: Inhibition, Construct, Reporter Assay, Luciferase, Control, Binding Assay, Negative Control, Amplification

Journal:

Article Title: Up-Regulation of Activating Transcription Factor-5 Suppresses SAP Expression to Activate T Cells in Hemophagocytic Syndrome Associated with Epstein-Barr Virus Infection and Immune Disorders

doi: 10.2353/ajpath.2008.080440

Figure Lengend Snippet: ATF5 exactly binds to site −305 to −296 and site −81 to −74 for suppressing SAP promoter activity. A: Predication of ATF5 binding sites on region −420 to −210 and region −210 to +11 of the SAP promoter. The three predicated sites were separately mutated (M1, M2, and M3) to identify crucial sites. B: Luciferase reporter constructs of the SAP promoter were analyzed for the LMP1-repressable region. Data were relative to control cells and expressed as the mean + SD of quadruplicate sample. C: EMSA was done using 10 μg of nuclear extract proteins from pSG5-H9 or LMP1-H9 cells. Biotin-labeled probes contained ATF5 binding site of regions −305 to −296 (top) and −81 to −74 (bottom) of SAP promoter. Unlabeled competitive DNA and mutated competitor (M; consisted of the M2 or M3 in A) were added as indicated. The anti-ATF5 antibody (A) could recognize the partial DNA binding domain bZIP of ATF5, and interfere the binding of labeled DNA. The anti-histone H1 (H) antibody was added as negative control. D: Binding on the region −305 to −296 was also shown in ATF5-overexpressed.H9 cells, but less in pSG5-H9 cells.

Article Snippet: Anti-LMP1 mouse monoclonal antibody (CS1-4; DAKO, Glostrup, Denmark), anti-ATF5 antibody (Imgenex), anti-SAP rabbit antibody, anti-α-actin goat antibody (Santa Cruz Biotechnology), and NF-κB inhibitor Bay11-7082 (Sigma) were used.

Techniques: Activity Assay, Binding Assay, Luciferase, Construct, Control, Labeling, Negative Control

Journal: Science signaling

Article Title: Sporadic activation of an oxidative stress-dependent NRF2–p53 signaling network in breast epithelial spheroids and premalignancies

doi: 10.1126/scisignal.aba4200

Figure Lengend Snippet: (A) Maximum-likelihood inference parameterization (lower) of a two-state distribution of transcript abundances for the gene cluster from microarray profiles (upper) of ECM-attached basal-like MCF10A-5E breast epithelial cells, randomly collected as 10-cell pools (n = 16) from 3D-cultured spheroids after 10 days, extracted from (20). Inferred expression frequencies are the maximum likelihood estimate with 90% confidence interval (CI). (B) Venn diagram summarizing the candidate transcription factors predicted from four different bioinformatics algorithms (data file S1). (C and D) Quantitative immunofluorescence of (C) hyperphosphorylated RB (pRB, an upstream proxy of active E2F1) and (D) NRF2 in 3D culture with ECM (upper), 2D culture (middle), and 2D culture with ECM (lower). Expression frequencies for a two-state lognormal mixture model (preferred over a one-state model by F test; p < 0.05) were calculated by nonlinear least squares of 60 histogram bins collected from n = 1100–1600 of cells quantified from 100–200 spheroids from two separate 3D cultures. For each subpanel, representative pseudocolored images are shown in the upper right inset and merged (magenta) with DAPI nuclear counterstain (blue) in the lower right inset. Scale bar is 10 μm.

Article Snippet: Lentiviruses were prepared in human embryonic kidney 293T cells (ATCC) by triple transfection of the viral vector with psPAX2 + pMD.2G (Addgene) and transduced into MCF10A-5E, MCF10DCIS.com, HCC1937, SUM159PT, MDA-MB-231, HCC1806, and MDA-MB-468 as previously described ( 25 ).

Techniques: Microarray, Cell Culture, Expressing, Immunofluorescence

Journal: Science signaling

Article Title: Sporadic activation of an oxidative stress-dependent NRF2–p53 signaling network in breast epithelial spheroids and premalignancies

doi: 10.1126/scisignal.aba4200

Figure Lengend Snippet: (A) Transcripts covarying with the median NRF2-associated fluctuation signature (Fig. 1A, upper; 20) measured by 10-cell RNA sequencing (45) of ECM-attached MCF10A-5E cells grown as 3D spheroids (n = 18 10-cell pools from GSE120261). Selected Gene Ontology enrichment analysis (green and purple) is shown for the transcripts with a Spearman correlation (ρ) greater than 0.5. The complete list of enrichments is available in file S2. (B) Quantitative immunofluorescence of NRF2 and p53 abundance in ECM-attached MCF10A-5E cells grown as 3D spheroids. Representative pseudocolored images for NRF2 (upper left) and p53 (middle left) are shown merged with DAPI nuclear counterstain (lower left). White arrows indicate concurrent NRF2 and p53 stabilization. Median-scaled two-color average fluorescence intensities are quantified (right) along with the log-scaled and background-subtracted mutual information (MI) with 90% CI for n = 1691 cells segmented from 50–100 spheroids from two separate 3D cultures. (C) Genetic perturbation of NRF2 by inducible shRNA knockdown (upper) and p53 by inducible expression of a FLAG-tagged carboxy terminal (residues 1–13, 302–390) dominant-negative p53 (DNp53, lower). NRF2 knockdown reduced NRF2 protein abundance to 22 ± 4% of control knockdown (fig. S3B). MCF10A-5E cells were treated with 1 μg/ml doxycycline for 72 (upper) or 24 (lower) hours and immunoblotted for NRF2 or FLAG with vinculin, tubulin, and p38 used as loading controls and p21 used to confirm efficacy of DNp53. The negative control for shNRF2 was an inducible shGFP, and the negative control for DNp53 was an inducible FLAG-tagged LacZ. (D) Abundance changes in the gene cluster after single and combined perturbations of NRF2 and p53. NQO1 was used as a control for efficacy of shNRF2, and CDKN1A shows efficacy of DNp53. MCF10A-5E cells with or without NRF2 knockdown or DNp53 were treated with 1 μg/ml doxycycline for 48 hours, grown as 3D spheroids for 10 days, and profiled for the indicated genes by quantitative PCR. Data are log2 geometric mean relative to the negative control (shGFP + FLAG-tagged LacZ), with asterisks indicating significant changes (left and middle columns) or interaction effects (right column) by two-way ANOVA of n = 8 independent 3D-cultured samples and a false-discovery rate of 5%. The complete set of transcripts in the gene cluster is shown in fig. S2C. (E) Dual inactivation of NRF2 and p53 causes synergistic proliferative suppression in MCF10A-5E 3D spheroids. Black arrows indicate proliferation-suppressed spheroids. Data are mean percentage of proliferation-suppressed spheroids ± s.e.m. of n = 8 independent 3D-cultured samples after 10 days. Statistical interaction between NRF2 and p53 (pint) was assessed by two-way ANOVA with replication. Scale bars are 20 μm (B) and 100 μm (E).

Article Snippet: Lentiviruses were prepared in human embryonic kidney 293T cells (ATCC) by triple transfection of the viral vector with psPAX2 + pMD.2G (Addgene) and transduced into MCF10A-5E, MCF10DCIS.com, HCC1937, SUM159PT, MDA-MB-231, HCC1806, and MDA-MB-468 as previously described ( 25 ).

Techniques: RNA Sequencing Assay, Immunofluorescence, Fluorescence, shRNA, Expressing, Dominant Negative Mutation, Negative Control, Real-time Polymerase Chain Reaction, Cell Culture

Journal: Science signaling

Article Title: Sporadic activation of an oxidative stress-dependent NRF2–p53 signaling network in breast epithelial spheroids and premalignancies

doi: 10.1126/scisignal.aba4200

Figure Lengend Snippet: (A) Quantitative immunofluorescence of NRF2 and p53 abundance in ECM-attached MCF10DCIS.com cells grown as 3D spheroids. Median-scaled two-color average fluorescence intensities are quantified along with the log-scaled and background-subtracted mutual information (MI) with 90% CI for n = 1832 cells segmented from 70–110 spheroids from two separate 3D cultures. (B) Common changes in transcript abundance identified by RNA sequencing of MCF10A-5E (5E) and MCF10DCIS.com (DCIS.com) cells grown as 3D spheroids with or without NRF2 knockdown. The negative control for shNRF2 was an inducible shGFP (5E) or shLacZ (DCIS.com). Data are log2-transformed Z-scores for genes detected at >5 transcripts per million from n = 4 biological replicates. Enriched gene sets for the BRCA1, ATM, and CHEK2 networks are indicated, with black denoting multiple enrichments. The complete list of enrichments is available in data file S3. (C) Quantification of rounded spheroids (circularity > 0.9) in 3D-cultured MCF10DCIS.com cells with or without NRF2 knockdown. The negative control for shNRF2 was an inducible shLacZ. (D) Quantification of large spheroids (size > e8.5 ~ 5000 μm2) in 3D-cultured MCF10DCIS.com cells with or without p53 disruption. The negative control for p53 constructs was an inducible FLAG-tagged LacZ. (E) Quantification of size and circularity in 3D-cultured MCF10DCIS.com cells with or without NRF2 knockdown, p53 disruption, or both. For (C) to (E), cells with or without inducible perturbations were treated with 1 μg/ml doxycycline for 48 hours, grown as 3D spheroids for 10 days, imaged by brightfield microscopy, and segmented. For (C) and (D), data are mean ± 90% bootstrap-estimated CI from n = 8 biological replicates, with p values by rank-sum test estimated by bootstrapping. For (E), data are mean ± s.e.m. of n = 8 biological replicates. Statistical interaction between NRF2 and p53 perturbations (pint) was assessed by two-way ANOVA with replication. Scale bars are 100 μm.

Article Snippet: Lentiviruses were prepared in human embryonic kidney 293T cells (ATCC) by triple transfection of the viral vector with psPAX2 + pMD.2G (Addgene) and transduced into MCF10A-5E, MCF10DCIS.com, HCC1937, SUM159PT, MDA-MB-231, HCC1806, and MDA-MB-468 as previously described ( 25 ).

Techniques: Immunofluorescence, Fluorescence, RNA Sequencing Assay, Negative Control, Transformation Assay, Cell Culture, Construct, Microscopy

Journal: Science signaling

Article Title: Sporadic activation of an oxidative stress-dependent NRF2–p53 signaling network in breast epithelial spheroids and premalignancies

doi: 10.1126/scisignal.aba4200

Figure Lengend Snippet: (A and B) NRF2 and p53 stabilization by oxidative stress compared to DNA double-strand breaks. MCF10A-5E cells were treated with 5 μM doxorubicin (double-strand breaks) or 200 μM H2O2 (oxidative stress) for the indicated time points, and NRF2 (magenta) or p53 (green) protein abundance was estimated by quantitative immunoblotting. Data are mean ± s.e.m. of n = 3 (A) or 4 (B) independent perturbations. (C) Endogenous oxidative stress association with NRF2 stabilization in 3D spheroids. MCF10A-5E cells stably expressing HyPer-2 (67) and mRFP1-NRF2 reporter (NRF2rep) were grown as 3D spheroids for 10 days and imaged by laser-scanning confocal microscopy. Representative pseudocolored images for HyPer-2 ratio (upper left) and mRFP1-NRF2 reporter (lower left) are shown. HyPer-2 ratios and mRFP1-NRF2 reporter fluorescence are quantified (right) along with the log-scaled mutual information (MI) with 90% CI for n = 605 cells segmented from 10–25 spheroids from four separate 3D cultures. (D) Suppression of endogenous NRF2–p53 coordination during 3D culture with the antioxidant Trolox. Representative pseudocolored images for NRF2 (upper left) and p53 (middle left) are shown merged with DAPI nuclear counterstain (lower left). White arrows indicate concurrent NRF2 and p53 stabilization. The log-scaled and background-subtracted MI (right) is shown with 90% CI estimated from n = 1000 bootstrap replicates. (E) Trolox interference with the synergistic proliferative suppression caused by dual inactivation of NRF2 and p53 in MCF10A-5E cells. Data are mean percentage of proliferation-suppressed spheroids ± s.e.m. of n = 8 independent 3D-cultured samples after 10 days. The overall effect of Trolox on spheroid size is shown in fig. S10. Statistical interaction between Trolox and NRF2–p53 (pint) was assessed by three-way ANOVA with replication. For (A) and (B), change in protein abundance over time was assessed by one-way ANOVA. For (D) and (E), MCF10A-5E cells cultured for 10 days in 3D with or without 50 μM Trolox supplemented every two days. Scale bars are 10 μm (C) and 20 μm (D).

Article Snippet: Lentiviruses were prepared in human embryonic kidney 293T cells (ATCC) by triple transfection of the viral vector with psPAX2 + pMD.2G (Addgene) and transduced into MCF10A-5E, MCF10DCIS.com, HCC1937, SUM159PT, MDA-MB-231, HCC1806, and MDA-MB-468 as previously described ( 25 ).

Techniques: Western Blot, Stable Transfection, Expressing, Confocal Microscopy, Fluorescence, Cell Culture

Journal: Science signaling

Article Title: Sporadic activation of an oxidative stress-dependent NRF2–p53 signaling network in breast epithelial spheroids and premalignancies

doi: 10.1126/scisignal.aba4200

Figure Lengend Snippet: (A) Transcripts per million for the indicated TNBC cell lines scaled to MCF10A cells from the NIH LINCS dataset (91). (B) NRF2–p53 mutual information (MI) and ROS tolerance for TNBC cell lines using the simulation strategy in Fig. 5B. (C to G) Quantification of mean spheroid area with or without NRF2 knockdown in 3D-cultured TNBC cells with higher simulated ROS tolerance and NRF2–p53 MI (C and D) and with lower simulated ROS tolerance and NRF2–p53 MI (E to G). (H) Transcripts per million for the TNBC lines in (A) scaled to clinical cases of TNBC in TCGA (35). (I) Simulated NRF2–p53 MI and ROS tolerance for TNBC tumors. Vertical lines indicate cases with high MI in the lower quartile of ROS tolerance. For (A) and (H), the clustered transcripts were used to adjust the initial conditions of the model simulations for each cell line and tumor. For (B) and (I), ROS tolerance was defined as the integrated intracellular H2O2 concentration in each cell line compared to that of MCF10A-5E cells in response to an increased ROS production rate as in Fig. 5B. For (C) to (G), TNBC cells with or without inducible NRF2 knockdown were treated with 1 μg/ml doxycycline for 72 hours, grown as 3D spheroids, imaged by brightfield microscopy, and segmented. Data are mean ± s.e.m. of n = 4–8 biological replicates. The difference between means was assessed by Student’s t test with Šidák correction for multiple-hypothesis testing, and the specific p53 mutation of each line is shown (lower left).

Article Snippet: Lentiviruses were prepared in human embryonic kidney 293T cells (ATCC) by triple transfection of the viral vector with psPAX2 + pMD.2G (Addgene) and transduced into MCF10A-5E, MCF10DCIS.com, HCC1937, SUM159PT, MDA-MB-231, HCC1806, and MDA-MB-468 as previously described ( 25 ).

Techniques: Cell Culture, Concentration Assay, Microscopy, Mutagenesis

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: Affymetrix probe list

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF is decreased in patients with NB and correlates with survival. A) HBEGF expression in the microarray meta-dataset in benign neuroblastic tumors (ganglioneuroma/ganglioneuroblastoma) or NB tumors. ****P < 0.0001 (Mann-Whitney U test). B) Immunofluorescence in neuroblastoma specimens for HBEGF (green). DAPI nuclear stain in blue. Original magnification, ×40. Scale bar, 50 μM. C) HBEGF expression in patients with NB by stage. P < 0.0001, Kruskal-Wallis test. **P < 0.01, ***P < 0.001, ****P < 0.0001 Mann-Whitney U test for intergroup comparisons. D) Event-free survival (EFS) in patients with NB with low (bottom 50%; red) and high (top 50%; blue) HBEGF expression in the GSE49710 dataset. E) HBEGF expression in the GSE49710 dataset in nonamplified (NA) or MYCN-amplified (Amp) NB tumors. ****P < 0.0001 (Mann-Whitney U test). Box plots are presented as median (horizontal bars) and interquartile range (boxes).

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Microarray, MANN-WHITNEY, Immunofluorescence, Staining, Amplification

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF promotes neuroblast differentiation in NB cells. A) Western blot for differentiation markers after 72 h of HBEGF treatment in BE2 and SK-N-AS. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. B) Quantification of NF160 densitometry normalized to β-actin from 3 independent Western blots (5Y, BE2) or 8 independent Western blots (SK-N-AS) after 72-h treatment with 1 ng/ml HBEGF and presented as means ± sem. P < 0.001 (1-way ANOVA). *P < 0.05, **P < 0.01, ****P < 0.0001, 1-sample Student’s t test. C) Representative phase-contrast images of BE2 cells after 72 h of treatment with HBEGF. Arrows identify long neurites. Original magnification, ×10. Scale bar, 100 μM. D) Quantification of neurite length using NeuronJ after 72 h of treatment with HBEGF from 3 independent experiments. P < 0.01 (1-way ANOVA). *P < 0.05, 1-sample Student’s t test. E) Western blot for differentiation markers after 72 h of HBEGF (0.5 or 1 ng/ml), FGF2 (1 or 10 ng/ml), or ATRA (1 or 10 μM). Densitometry for NF160 normalized to β-actin is shown as the percentage of control. F) Western blot for differentiation markers after 72 h HBEGF (0, 0.25, 0.5, 0.75, 1, 2 ng/ml) and a neutralizing HBEGF antibody (nAb; 0.0075, 0.015, 0.03, 0.05, 0.1, or 0.5 μg/ml). Densitometry for NF160 normalized to β-actin is shown as the percentage of control. G) Western blot for β3-tubulin and HBEGF in SHEP stably expressing an NTC shRNA or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for β3-tubulin normalized to β-actin is shown as the percentage of control. H) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right).

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Microarray

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: Schwannian stroma-derived HBEGF promotes neuroblast differentiation. A) Immunofluorescence in NB specimens using HBEGF (green) and S100 schwannian stroma (red) antibodies. DAPI nuclear stain in blue. Original magnification, ×40. Scale bar, 50 μM. B) Western blot for differentiation markers in 5Y after 72 h of coculture or treatment with conditioned medium from SHEP stably expressing an NTC shRNA construct or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for NF160 normalized to β-actin is shown as the percentage of control.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Immunofluorescence, Staining, Western Blot, Stable Transfection, Expressing, shRNA, Construct, Control

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HSPGs and EGFR interact to promote HBEGF-mediated neuroblast differentiation. A) Western blots for differentiation markers after 72-h treatment with 10 ng/ml soluble (s)TβRIII or sCD44 or 100 ng/ml sGPC1, sGPC3, or sSDC3 in the absence or presence of 0.5 ng/ml HBEGF in 5Y cells. B) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, a dosecourse of HBEGF in SK-N-AS after 96-h TβRIII knockdown. C) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, 0.5 μg/ml heparin, ODSH, 2-O desulfated heparin (2DES), 6-O desulfated heparin (6DES), or N desulfated heparin (NDES) in the absence or presence of 0.5 ng/ml HBEGF in SK-N-AS. D) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, EGFR inhibitors for 72 h followed by 48 h of 1 ng/ml HBEGF treatment. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. E) In situ proximity ligation assay in SK-N-AS after 5 min of treatment with 1 ng/ml HBEGF or 1 ng/ml EGF. Original magnification, ×40. Scale bars, 50 μM. Normalized TβRIII/EGFR complexes per cell (75–100 cells/condition) from 6 independent experiments. P < 0.001 (1-way ANOVA). *P < 0.05, ****P < 0.0001, 1-sample Student’s t test. F) Analysis of event-free survival in the GSE49710 dataset stratified by the top and bottom 50% for HBEGF, then TGFBR3, then EGFR expression (left) and analysis of event-free survival stratified by the top and bottom 12.5% for HBEGF, then TGFBR3, then EGFR expression (middle) compared with stratification by MYCN amplification status (right). NA, nonamplified.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control, Knockdown, In Situ, Proximity Ligation Assay, Expressing, Amplification

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF induces neuroblast differentiation via ERK and STAT3 signaling and up-regulation of ID1. A) Western blot in SK-N-AS for phosphorylated and total STAT3 or ERK1/2 after 72 h treatment with 1 ng/ml HBEGF. Densitometry for phosphorylated STAT3 or phosphorylated ERK1/2 normalized to β-actin is shown as the percentage of control. B) Western blot for ID1 in BE2 and SK-N-AS after 72 h treatment with a dosecourse of HBEGF. Densitometry for ID1 normalized to β-actin is shown as the percentage of control. C) Western blot for ID1 in SHEP stably expressing an NTC shRNA or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for ID1 normalized to β-actin is shown as the percentage of control. D) Western blot in SK-N-AS after 96 h ID1 knockdown and 72 h HBEGF treatment. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. E) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right). F) Western blot for differentiation markers and ID1 after 24 h cotreatment with 1 ng/ml HBEGF and the indicated doses of U0126 or CI-1040. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. G) Western blot for differentiation markers after 72 h expression of an empty-vector control (EV) or a dominant negative STAT3 (DN STAT3) construct and 48 h treatment with 1 ng/ml HBEGF or 48 h treatment with ruxolitinib and 24 h treatment with 1 ng/ml HBEGF. Densitometry for NF160 normalized to β-actin is shown as the percentage of control.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Microarray, Plasmid Preparation, Dominant Negative Mutation, Construct

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF suppresses neuroblast proliferation. A) Proliferation index from 3 (5Y) or 4 (SK-N-AS, BE2) replicates (means ± sem) of thymidine incorporation after HBEGF treatment for 24 h (SK-N-AS), 48 h (5Y), or 72 h (BE2), normalized to untreated control. P < 0.0001 (1-way ANOVA). **P < 0.01, ***P < 0.001 (1-sample Student’s t test). B) Western blot for p21 after 72 h of HBEGF treatment in BE2 and SK-N-AS. Densitometry for p21 normalized to β-actin is shown as the percentage of control. C) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right). D) SK-N-AS NTC and SK-N-AS shHBEGF#1 subcutaneous xenograft. Tumors were measured at d 19 using calipers, and this measurement was used to calculate the fold change in tumor growth after 21, 24, 26, and 31 d. **P < 0.01, Kruskal-Wallis to compare curves.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Control, Western Blot, Microarray