Journal: Science advances

Article Title: Matrix-bound nanovesicles alleviate particulate-induced periprosthetic osteolysis.

doi: 10.1126/sciadv.adn1852

Figure Lengend Snippet: Fig. 3. MBVs mitigate RANKL-induced osteoclast formation and functions by suppressing NF-κB pathway in vitro. RAW 264.7 cells were challenged with RANKL and various concentrations of MBV (1× 1010 to 2× 1010 particles/ml, MBV versus cells ratio 1× 104 to 2 × 104 particles/ml per cell), 1 day for osteoclast differentiation and activity– related gene RT-qPCR (A and B; RANKL, 50 ng/ml), 3 days for osteoclast differentiation and activity–related protein Western blot (C; RANKL, 50 ng/ml), and 5 days for TNF-α concentration in conditional media (D; RANKL, 30 ng/ml; MBV, 5× 109 particles/ml). (A) Osteoclast differentiation regulators (NFATc1 and DC-STAMP) relative expression (2−△△T) were measured by RT-qPCR (values = means ± SD; n = 9; significant differences, *P < 0.05). (B) Osteoclast activity–related genes (cathepsinK, c-Src, MMP-9, and β3-integrin) relative expressions (2−△△T) were measured by RT-qPCR (values = means ± SD; n = 9; significant differences, *P < 0.05). (C) Osteoclast differentiation–related protein (NFATc1) and osteoclast activity proteins (c-Src and cathepsin K) relative expression was measured by Western blot (values = means ± SD; n = 3; significant differ- ences, *P < 0.05). (D) TNF-α in conditional media from Raw 264.7 cells cocultured with RANKL with or without MBV for 5 days was determined by ELISA (values = means ± SD; n = 3; significant differences, *P < 0.05). (E) RAW 264.7 cells were challenged with RANKL (50 ng/ml) and MBV (2 × 1010 particles/ml) from 0 to 60 min. NF-κB pathway proteins were determined by Western blot. Representative Western blotting images of the effects of MBV on p-p65, p65, and IĸB-α. Quantification of the ratios of band intensity of p-p65/p65 and IĸBα relative to β-actin expression (values = means ± SD; n = 3; significant differences, *P < 0.05).

Article Snippet: Then, membranes were blocked with 5% nonfat milk in tris- buffered saline containing 0.1% Tween- 20 (TBST) for 1 hour at room temperature, and subsequently probed with 1:1000 dilution primary antibodies NFATc1 (8032S,Cell Signaling Technology, USA), cathepsin K (57056S,Cell Signaling Technology, USA), c- Src (25978- 1- AP, Proteintech, USA), phospho- p65 (3033S, Cell Signaling Technology, USA), p65 (8242S, Cell Signaling Technology, USA), IκBα (4812S, Cell Signaling Technology, USA), and βactin (4970T, Cell Signaling Technology, USA) in TBST and 1% bovine serum albumin (BSA) overnight at 4°C.

Techniques: In Vitro, Activity Assay, Quantitative RT-PCR, Western Blot, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Matrix-bound nanovesicles alleviate particulate-induced periprosthetic osteolysis.

doi: 10.1126/sciadv.adn1852

Figure Lengend Snippet: Fig. 7. Schematic illustration of the mechanism by which MBVs reduce particulate-induced osteolysis. MBVs were derived from a porcine urinary bladder extracel- lular matrix (UBM) bioscaffold. In vitro, MBVs reduce monocyte/macrophage differentiation to osteoclast by suppressing the NF-κB pathway and its downstream NFATc1, DC-STAMP, c-Src, and cathepsin K expression. In vivo, MBVs alleviate UHMWPE particle-induced osteolysis in a murine calvarial osteolysis model.

Article Snippet: Then, membranes were blocked with 5% nonfat milk in tris- buffered saline containing 0.1% Tween- 20 (TBST) for 1 hour at room temperature, and subsequently probed with 1:1000 dilution primary antibodies NFATc1 (8032S,Cell Signaling Technology, USA), cathepsin K (57056S,Cell Signaling Technology, USA), c- Src (25978- 1- AP, Proteintech, USA), phospho- p65 (3033S, Cell Signaling Technology, USA), p65 (8242S, Cell Signaling Technology, USA), IκBα (4812S, Cell Signaling Technology, USA), and βactin (4970T, Cell Signaling Technology, USA) in TBST and 1% bovine serum albumin (BSA) overnight at 4°C.

Techniques: Derivative Assay, In Vitro, Expressing, In Vivo

Journal: Cancer Research

Article Title: Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

doi: 10.1158/0008-5472.can-14-1592

Figure Lengend Snippet: Figure 2. Inhibition of NFATc1 in MC38Met cells. Effect of control RNAi (siScr) or NFATc1-specific RNAi on specific NFAT mRNA species: NFATc1 (A), NFATc2 (B), NFATc3 (C). D, the effect of NFATc1 siRNA versus scrambled control si-Scr on NFAT family protein levels in MC38Met cells, and steady state levels in both MC38Par and MC38Met cells with Ramos cell lysate positive control in the right hand lane. E, relative rates of invasion for cells shown in A–D . , P < 0.0005; , P < 0.00005.

Article Snippet: For preparing stable overexpressing cells, mouse wildtype NFATc1 (Addgene plasmid 11101; ref. 20) was cloned into vector pGP-Lenti3 (GenBank Accession no. JX861384) between unique XbaI and BamHI sites.

Techniques: Inhibition, Control, Positive Control

Journal: Cancer Research

Article Title: Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

doi: 10.1158/0008-5472.can-14-1592

Figure Lengend Snippet: Figure 3. NFATc1 expression is associated with cell invasion in human colon cancer cells. A, Western blot analysis showing NFATc1 levels in vector- transfected and NFATc1-transfected HT29 colon cancer cells. B, representative live cell measures after a 24-hour growth period using pools of HT29 cells shown in A. ns, not significant. C, relative rates of invasion through Matrigel at 24 hours (, P < 0.05) of cells depicted in A. D, Western blot analysis showing protein expression of NFATc1-c3 family members in HCT116 cells treated with either control RNAi (si-Scr) or NFATc1-specific RNAi (si-NFATc1). E, representative measures of HCT116 cells shown in D . F, relative measures of live cell growth of HCT116 control (Scr) and NFATc1 knockdown (siNFATc1) after 24 hours in culture. ns, not significant. G, relative rate of HCT116 cell invasion through Matrigel shown in D–F (representative experiment, P < 0.005).

Article Snippet: For preparing stable overexpressing cells, mouse wildtype NFATc1 (Addgene plasmid 11101; ref. 20) was cloned into vector pGP-Lenti3 (GenBank Accession no. JX861384) between unique XbaI and BamHI sites.

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Knockdown, Relative Rate

Journal: Cancer Research

Article Title: Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

doi: 10.1158/0008-5472.can-14-1592

Figure Lengend Snippet: Figure 4. Evidence for a dominant set of NFATc1 target genes. A, expression of eight putative target mRNA species significantly associated with NFATc1 expression inMC38Met cellsfollowing FK506 treatment (relative toDMSO control, n¼ 4, significance is determined by one-sample t testagainstequivalence or ratio ¼ 1 as shown by vertical line). B, expression of eight mRNA species significantly associated with NFATc1 expression in HCT116 cells following treatment with NFATc1-specific RNAi (relative to siSCR, n ¼ 4, significance is determined by one-sample t test against equivalence or ratio ¼ 1 as shown by vertical line). C, quantification of NFATc1 pulldown on putative target gene-specific promoter binding elements relative to IgG control (ASPN mRNA species undetectable, not shown). Not significant, P > 0.05; , P < 0.05; , P < 0.005; , P < 0.0005; , P < 0.00005.

Article Snippet: For preparing stable overexpressing cells, mouse wildtype NFATc1 (Addgene plasmid 11101; ref. 20) was cloned into vector pGP-Lenti3 (GenBank Accession no. JX861384) between unique XbaI and BamHI sites.

Techniques: Expressing, Control, Binding Assay

Journal: Cancer Research

Article Title: Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

doi: 10.1158/0008-5472.can-14-1592

Figure Lengend Snippet: Figure 5. An NFAT-driven transcriptional program is correlated with poor outcomes in stage II and III colorectal cancer patients. A and B, NFATc1 signature gene expression patterns in stages II and III colorectal cancer specimens in the Vanderbilt Medical Center (GSE 38832; A) and the Moffitt Cancer Center (GSE17536; B) datasets. The top three rows indicate whether or not a disease-specific death event or a recurrence event was recorded in follow-up (black, no event; red, event; white, not available), respectively. The fourth row indicates the cancer stage (green, stage II; dark blue, stage III). C–E, OS, DSS, and DFS analyses based on combined Vanderbilt and Moffitt datasets.

Article Snippet: For preparing stable overexpressing cells, mouse wildtype NFATc1 (Addgene plasmid 11101; ref. 20) was cloned into vector pGP-Lenti3 (GenBank Accession no. JX861384) between unique XbaI and BamHI sites.

Techniques: Gene Expression

Journal: Cancer Research

Article Title: Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

doi: 10.1158/0008-5472.can-14-1592

Figure Lengend Snippet: Figure 6. Overexpression of NFATc1 in MC38Par cells increases tumor incidence and liver metastases. A, Western blot analysis showing NFAT proteins in MC38Par cells transfected with eitherempty vector (VEC) or NFATc1. b-actin was used asa loading control andRamos extract aspositivecontrol. B, analysis of NFATc1 mRNA in MC38Par cells shown in A. C, rates of trans-endothelial invasion for MC38Par cells shown in A and B. Individual replicate wells from a representative experimentare plotted with the mean and theSEM (bars and whiskers).D, representative mice from splenic metastasis model (n¼ 12-14/group) using MC38Par cells shown in A–D. E, bioluminescence of mice injected with MC38Par cells shown in A–D at day 14 postinjection. F, incidence of liver metastases for mice injected at day 14. G, liver weight to body weight ratio in mice injected with either MC38Par shown in A–F cells at day 14 postinjection. , P < 0.02; , P ¼ 0.0004; , P < 0.0001.

Article Snippet: For preparing stable overexpressing cells, mouse wildtype NFATc1 (Addgene plasmid 11101; ref. 20) was cloned into vector pGP-Lenti3 (GenBank Accession no. JX861384) between unique XbaI and BamHI sites.

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Control, Injection

Journal: Cancer Research

Article Title: Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

doi: 10.1158/0008-5472.can-14-1592

Figure Lengend Snippet: Figure 7. Knockdown of NFATc1 in MC38Met cells decreases tumor incidence and liver metastases. A, Western blot analysis showing NFAT proteins in MC38MetþshCtrl and MC38MetþshNFATc1 cell lines. b-actin was used as a loading control and Ramos cell extract as positive control. B, analysis of NFATc1 specific mRNA in cells shown in A. C, rates of trans-endothelial invasion for cells are shown in A and B. Individual replicate wells from a representative experiment are plotted with the mean and the SEM (bars and whiskers). D, representative (n ¼ 14-15 mice/group) bioluminescence image corresponding day 21. E, summary- of bioluminescence signal quantified at day 21. F, summary incidence of liver metastases. G, liver metastases measured by liver weight to body weight ratio at 21 days postinjection. , P < 0.03; , P < 0.001; , P < 0.0001.

Article Snippet: For preparing stable overexpressing cells, mouse wildtype NFATc1 (Addgene plasmid 11101; ref. 20) was cloned into vector pGP-Lenti3 (GenBank Accession no. JX861384) between unique XbaI and BamHI sites.

Techniques: Knockdown, Western Blot, Control, Positive Control

Journal: Journal of Biological Chemistry

Article Title: p38α Protein Negatively Regulates T Helper Type 2 Responses by Orchestrating Multiple T Cell Receptor-associated Signals

doi: 10.1074/jbc.m112.355594

Figure Lengend Snippet: FIGURE 4. p38 expression is compromised during T helper cell differen- tiation. A, naïve CD4 T cells were activated with immobilized anti-CD3 (2 g/ml) and anti-CD28 (1 g/ml) and cultured under Th0 conditions for the indicated times. Protein levels of p38 and NFATc1 were detected by immu- noblotting. B, naïve CD4 T cells were pretreated with bafilomycin A1 (100 ng/ml) and MG132 (20 M), respectively, then activated by anti-CD3 and anti- CD28. p38 levels were measured by immunoblotting. C, naïve CD4 T cells were cultured as panel A. Total RNA was extracted, and mRNA levels of p38 were detected by real-time PCR. p38 expression relative to -actin is shown as the mean S.D. from triplicate wells. *, p 0.05. Data are representative of three independent experiments.

Article Snippet: Mouse 33216 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 40 • SEPTEMBER 28, 2012 by guest on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from NFATc1 shRNA sequences were obtained from Origene and were cloned into pMKO.1 GFP vector.

Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: p38α Protein Negatively Regulates T Helper Type 2 Responses by Orchestrating Multiple T Cell Receptor-associated Signals

doi: 10.1074/jbc.m112.355594

Figure Lengend Snippet: FIGURE 5. Altered NFAT homeostasis is required for enhanced Th2 differentiation due to p38 deficiency. A, naïve CD4 T cells were collected from p38/ and p38/ mice. Total cell lysates were prepared and analyzed by immunoblotting for NFATc1, NFATc2, JunB, Fyn, and p38 expression. B, CD4 TcellsweretreatedwithSB203580(SB;10M),andproteinlevelsofNFATc1andNFATc2weredetectedbyimmunoblotting.C,naïveCD4Tcellsfromp38/ and p38/ mice were stimulated with anti-CD3 and anti-CD28 for 48 h. Total protein was prepared, and expression of NFATc1 and NFATc2 was measured by immunoblotting. D, CD4 T cells from p38/ and p38/ mice were treated as in panel C. Nuclear protein was extracted, and protein levels of NFATc1 and P65 RelA were detected by immunoblotting. E, CD4 T cells were infected with control or NFATc1 shRNA retrovirus. Protein levels of sorted GFP cells were measured by immunoblotting. F, CD4 T cells from p38/ and p38/ mice were “spininfected” with control or NFATc1 shRNA retrovirus as panel E. Sorted GFP cells were further cultured under Th0 conditions, and percentages of IL-4 cells were analyzed by flow cytometry. G, shown is a quantitative analysis of the average percentage of IL-4 cells (n 3–4). *, p 0.05. NS, not significant. Data are the mean S.D. Signals in A–E were quantified by densitometric scanning.

Article Snippet: Mouse 33216 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 40 • SEPTEMBER 28, 2012 by guest on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from NFATc1 shRNA sequences were obtained from Origene and were cloned into pMKO.1 GFP vector.

Techniques: Western Blot, Expressing, Infection, Control, shRNA, Cell Culture, Flow Cytometry