Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4 + T cells. (A) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4 + T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4 + T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).

Article Snippet: Anti–human phospho NFATc1 (S172; clone 679340) was purchased from R&D Systems.

Techniques: Activation Assay, Expressing, Purification, Staining, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: MAPK-cascade activated p38 inhibits functions distal to alternatively activated p38. (A) Purified CD4 + T cells were cultured overnight without stimulation, and then treated with either UV (50 J/m 2 or 100 J/m 2 or 200 J/m 2 ), 0.6 M sorbitol for 30 min, or PMA (10 ng/ml) and ionomycin (1 µg/ml) for 1 h. Cell lysates were immunoblotted with anti-phospho-p38. The results are representative of three independent experiments. (B) Purified CD4 + T cells were treated with either 0.6 M sorbitol for 30 min, 50 J/m 2 UV, or PMA and ionomycin were stimulated with anti-CD3/CD28 for 24 h. The expression of Nfatc1 and Irf4 was determined by quantitative real-time PCR. Results are the mean ± SEM of three independent experiments. *, P < 0.05 (unpaired two-tailed Student’s t test). (C) Cells were treated as in B, and IRF4 expression was determined by Western blot. The results are representative of two independent experiments. (D) Freshly purified T cells were stimulated or not with anti-CD3/CD28 for 48 h. Cytosolic and nuclear fractions were separated in a low percentage SDS-PAGE (8%) gel and immunoblotted for NFATc1. The results are representative of three independent experiments. (E) CD4 + T cells were stimulated for 48 h with anti-CD3/CD28, and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min, rested for 1 h, then immunoblotted for NFATc1 in cytosolic and nuclear fractions. Results are representative of three independent experiments. (F) Purified CD4 + T cells were treated or not with sorbitol for 30 min and stimulated with anti-CD3/CD28 for 48 h, and then immunoblotted for NFATc1 in cytosolic and nuclear fractions. The results are representative of three independent experiments. (G) Jurkat T cells were stimulated for 48 h with an agonistic anti-TCR antibody (C305), and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min. After 1 h without further stimulation, cytosolic fractions were immunoblotted for phospho-NFATc1 (p-NFATc1). The results are representative of three independent experiments. (H) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in in vitro kinase buffer. After 1 h, recombinant ATF2 and 10 µCi [ 32 P]ATP were added for 30 min before separation on SDS-PAGE and PhosphorImager analysis. The phosphorylation state of p38 Y323 was determined by immunoblotting. The results are representative of three independent experiments. (I) Recombinant p38 was activated with Zap70, MKK6, or buffer alone and an in vitro kinase assay using recombinant NFATc1 as substrate was performed as in (H). The results are representative of two independent experiments. (J) Recombinant p38 was activated with MKK6 or buffer alone in in vitro kinase buffer. Active JNK1 was kept in in vitro kinase buffer. After 1 h, recombinant NFATc1 was added and incubated for an additional hour before separation on SDS-PAGE and immunoblotting with an antibody specific for NFATc1 phosphorylated on residue S172. The results are representative of two independent experiments. (K) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in an in vitro kinase assay with recombinant NFATc1 as the p38 substrate, as in I. Samples were separated on a low percentage SDS-PAGE (8%) and immunoblotted for NFATc1 phosphorylated on residue S172. The results are representative of three independent experiments.

Article Snippet: Anti–human phospho NFATc1 (S172; clone 679340) was purchased from R&D Systems.

Techniques: Purification, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, SDS Page, Recombinant, In Vitro, Phospho-proteomics, Kinase Assay, Incubation, Residue

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Regulation of initial VV organization by Foxc2 and Nfatc1. (A) Colocalization of Foxc2 (red) and Nfatc1 (green) in VFCs at P0. Foxc2 and Nfatc1 localization are also shown separately. n = 4. Bar, 20 µm. (B–E) The proportion of VVs identified at stage 0 (white) and stage 1 (gray) at P0 after deletion of Foxc2 (B) or Ppp3r1 (CnB1; C) from E15, or treatment with cyclosporin from E17 (D), or with combined Foxc2 deletion and cyclosporin treatment (E). The number of VVs analyzed for each condition is indicated above each bar. ***, P < 0.0005, χ 2 . (E) Localization of Prox1 (red) is shown at P0 after combined Foxc2 deletion and cyclosporin treatment. Bar, 20 µm.

Article Snippet: Antibodies were raised in rabbit to Cx43 (3512; Cell Signaling Technology), Cx37 (CX37A11; Alpha Diagnostics International), Prox1 (11-002P; AngioBio), cleaved caspase 3 (9661; Cell Signaling Technology), Ki-67 (ab15580; Abcam), sheep to Foxc2 (AF6989; R&D Systems), goat to Nfatc1 (AF5640; R&D Systems), Prox1 (AF2727; R&D Systems), and GFP (ab6658; Abcam) rat to PECAM1 (BD clone MEC 13.3) and mouse to α-SMA (clone 1A4 conjugated to Cy3 [Sigma-Aldrich] or FITC [Abcam]).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Maturation of leaflets and commissures. (A) Immunolocalization at P6 of Nfatc1 (green) and Prox1 (red) in VV of WT mice administered control solvent or cyclosporin ( n = 5 versus 8 VVs, P < 0.05, χ 2 ). (B) Immunolocalization of Foxc2 (green) and Prox1 (red) in VVs of mice of the indicated genotypes. n ≥ 6 per condition. (C and D) Quantification of leaflet length and stage of VV development in mice of the indicated genotypes. Error bars represent means ± SEM. t test for leaflet length. (C, D, G, H, and I) Colors represent developmental stage (see key). The number of VVs analyzed for each condition is given above each bar. *, P < 0.05; **, P < 0.005 (χ 2 for developmental stage). (E) Immunolocalization of Cx37 (arrowheads) and Cx43 in VV leaflets at P6. n ≥ 4. (F) Localization of Prox1, Foxc2, and SMA at P6 in Gja4 −/− vein. n ≥ 6 (G and H) Stages of VV development reached in mice of the indicated genotypes. (I) Localization of Prox1, GFP, and SMA in Gjc2 GFP/GFP KO reporter vein at P2 ( n = 4), and stages of VV development reached in mice of the indicated genotypes. (J) Localization of Cx47-expressing VV cells (arrowheads) of Gjc2 GFP/+ reporter mice at P6. n = 4. Multichannel images are reproduced in supplementary data. Bars, 20 µm. (A–J) Blue stain is PECAM1.

Article Snippet: Antibodies were raised in rabbit to Cx43 (3512; Cell Signaling Technology), Cx37 (CX37A11; Alpha Diagnostics International), Prox1 (11-002P; AngioBio), cleaved caspase 3 (9661; Cell Signaling Technology), Ki-67 (ab15580; Abcam), sheep to Foxc2 (AF6989; R&D Systems), goat to Nfatc1 (AF5640; R&D Systems), Prox1 (AF2727; R&D Systems), and GFP (ab6658; Abcam) rat to PECAM1 (BD clone MEC 13.3) and mouse to α-SMA (clone 1A4 conjugated to Cy3 [Sigma-Aldrich] or FITC [Abcam]).

Techniques: Control, Solvent, Expressing, Staining

Journal: Stem cells international

Article Title: Aucubin Impeded Preosteoclast Fusion and Enhanced CD31 hi EMCN hi Vessel Angiogenesis in Ovariectomized Mice.

doi: 10.1155/2022/5226771

Figure Lengend Snippet: Figure 3: Aucubin increases the number of preosteoclasts in vitro. (a) Cell viability of RAW264.7 cells cultured with different concentrations of aucubin (0, 1 μM, and 5 μM) was analyzed via a CCK-8 assay. (b–e) Relative mRNA expression levels of NFATc1, c-FOS, DC-STAMP, and ATP6V0D2 were analyzed. (f) Representative images of TRAP staining on day 6. Scale bar: 200 μm. (g) Quantification of osteoclasts (OCs) and preosteoclasts (POCs) on day 6. (h–j) Protein levels of c-Fos and NFATc1 were analyzed by WBs. (k) IF staining was performed to observe the location of NFATc1. Scale bar: 50 μm. (l) The quantification of the relative NFATc1 fluorescence intensity in different groups. ∗P < 0:05 compared to the control group; #P < 0:05 compared to the Rankl group.

Article Snippet: The membranes were stained with primary antibodies against GAPDH (1 : 10000), PDGF-BB (1 : 1000), cathepsin K (1 : 1000), c-fos (1 : 1000), NFATc1 (1 : 1000), p-JNK (1 : 500), JNK (1 : 1000), p-p38 (1 : 1000), p38 (1 : 1000), p-p65 (1 : 1000), p65 (1 : 1000), p-IKBα (1 : 1000), or IKBα (1 : 1000) at 4°C for 8 h. Finally, the membranes were incubated with secondary antibodies (1 : 20,000, Rockland, USA) for 1 hour at room temperature.

Techniques: In Vitro, Cell Culture, CCK-8 Assay, Expressing, Staining, Control