Journal: Chinese Journal of Cancer

Article Title: Expression and unique functions of four nuclear factor of activated T cells isoforms in non-small cell lung cancer

doi: 10.5732/cjc.010.10156

Figure Lengend Snippet: Expression of NFAT proteins and CaN in non–small cell lung cancer (immunohistochemistry). A, negative staining of NFAT1; B–E, positive staining of NFAT1, NFAT2, NFAT3, and NFAT4, respectively; F, positive staining of CaN. Bars , 100 µm.

Article Snippet: Mouse anti-human NFAT2 and mouse anti-human NFAT4 antibodies were purchased from Santa Cruz, Inc. Rabbit anti-human antibodies were purchased from Canadian Chemicom International, Inc. PV9000 and DAB reagents were purchased from Beijing Zhongshan Goldenbridge Biotechnology, Ltd.

Techniques: Expressing, Immunohistochemistry, Negative Staining, Staining

Journal: Chinese Journal of Cancer

Article Title: Expression and unique functions of four nuclear factor of activated T cells isoforms in non-small cell lung cancer

doi: 10.5732/cjc.010.10156

Figure Lengend Snippet: Association between NFAT1 and NFAT4 expression and clinicopathologic characteristics in 159 patients with non–small cell lung cancer

Article Snippet: Mouse anti-human NFAT2 and mouse anti-human NFAT4 antibodies were purchased from Santa Cruz, Inc. Rabbit anti-human antibodies were purchased from Canadian Chemicom International, Inc. PV9000 and DAB reagents were purchased from Beijing Zhongshan Goldenbridge Biotechnology, Ltd.

Techniques: Expressing

Journal: Chinese Journal of Cancer

Article Title: Expression and unique functions of four nuclear factor of activated T cells isoforms in non-small cell lung cancer

doi: 10.5732/cjc.010.10156

Figure Lengend Snippet: Correlation between the expressions of NFAT4 and CaN in non–small cell lung cancer

Article Snippet: Mouse anti-human NFAT2 and mouse anti-human NFAT4 antibodies were purchased from Santa Cruz, Inc. Rabbit anti-human antibodies were purchased from Canadian Chemicom International, Inc. PV9000 and DAB reagents were purchased from Beijing Zhongshan Goldenbridge Biotechnology, Ltd.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

doi: 10.1084/jem.20131272

Figure Lengend Snippet: NFAT4 is selectively activated by ectopic expression of GRA6. (A) 293T cells were transfected with the NFAT-dependent luciferase reporter together with indicated expression vectors. Luciferase activities in the presence or absence of CAMLG were expressed as indicated above. Error bars represent means ± SD of triplicates. (B) 293T cells were transfected with the NFAT-dependent luciferase reporter together with indicated expression vectors. Luciferase activities in the presence or absence of PMA/ionophore were expressed as indicated above. Error bars represent means ± SD of triplicates. (C) Lysates of 293T cells transiently transfected with HA-tagged CAMLG were immunoprecipitated with the indicated Abs and subjected to Western blotting. (D) Lysates of 293T cells transiently cotransfected with Flag-tagged indicated NFAT vectors and/or HA-tagged CAMLG were immunoprecipitated with the indicated Abs and subjected to Western blot. (E) Quantitative RT-PCR analysis was performed using cDNA reversely transcribed from RNA extracted from 293T cells stably expressing shRNA for control or NFAT1-4. Relative mRNA levels of indicated genes compared with the GAPDH level were shown in the y axis. Error bars represent means ± SD of triplicates. (F) 293T cells stably expressing shRNA for human NFAT1-4 or control were transfected with the NFAT-dependent luciferase reporter together with empty or GRA6 expression vectors. Error bars represent means ± SD of triplicates. (G) MEFs stably expressing RFP-tagged NFAT4 were transfected with empty or Flag-tagged GRA6 or GRA15 expression vectors. 24 h after transfection, the cells were fixed and stained with mouse anti-Flag, and then Alexa Fluor 488–conjugated anti–mouse IgG (green) or DAPI (blue). Bars, 5 µm. *, P < 0.001, determined by unpaired Student’s t test in A or B. Data are representative of three (A and F) and two (B–E and G) independent experiments.

Article Snippet: Antibodies against CAMLG, NFAT2, NFAT4, HA-probe, and anti-Actin were from Santa Cruz Biotechnology, Inc. Anti-Flag, PMA, and calcium ionophore (A23187) were obtained from Sigma-Aldrich.

Techniques: Expressing, Transfection, Luciferase, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Stable Transfection, shRNA, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

doi: 10.1084/jem.20131272

Figure Lengend Snippet: NFAT4 activation by T. gondii infection is dependent on GRA6. (A) MEFs stably expressing RFP-NFAT4 were uninfected or infected with T. gondii (moi = 5), fixed at 6 h after infection, and stained with DAPI. Bars, 5 µm. (B) Structure of the GRA6 gene, targeting vector, and predicted disrupted gene. Black boxes denote the exon. Restriction enzymes: B, BamHI. (C) Southern blot analysis of offspring from WT or two lines of GRA6 -deficient parasites. Total genomic DNA was extracted from parasites, digested with BamHI, electrophoresed, and hybridized with the radiolabeled probe indicated in B. Southern blotting gave a single 2.4-kb band for WT and a 6.4-kb band for the disrupted locus. (D) Quantitative RT-PCR analysis was performed using cDNA reversely transcribed from RNA extracted from WT and gra6-ko parasites. Relative mRNA levels of GRA6 compared with the α-tubulin level were shown in the y axis. Error bars represent means ± SD of triplicates. N.D., not detected. (E) Plaque assays. MEF monolayers were infected with WT or two independent gra6-KO parasites (#1 and #2), and then fixed after 8 d and stained with crystal violet. (F) Total number of WT or gra6-KO parasites infecting in MEFs were measured by luciferase assay after indicated hours after infection. Error bars represent means ± SD of triplicates. (G) Intracellular growth assays. Replication was analyzed 24 h after infection. Error bars represent means ± SD of triplicates. (H) Targeting vector complementing GRA6 and harboring the endogenous promoter. Black boxes denote the exon. Blue and red primers were used to measure mRNAs for GRA6 and a region specifically derived from the targeting vector, respectively. (I) Quantitative RT-PCR analysis was performed using cDNA reversely transcribed from RNA extracted from WT, gra6-KO, or KO+GRA6 parasites. Relative mRNA levels of GRA6 compared with the α-tubulin level were shown in the y axis. The results of qRT-PCR using primers blue and red were shown in left and right, respectively. Error bars represent means ± SD of triplicates. (J and K) MEFs stably expressing RFP-NFAT4 were infected with WT, gra6-KO, or gra6-KO parasites expressing GRA6 (KO+GRA6) T. gondii (moi = 5), fixed at 6 h after infection, and stained with DAPI. Bars, 5 µm. The representative images are shown in J and mean nuclear RFP signal was measured in K. The y axis represents the mean nuclear RFP signal (NFAT4) intensity over all the cells. *, P < 0.001 (WT vs. gra6-KO); **, P < 0.001 (gra6-KO vs. KO+GRA6), determined by unpaired Student’s t test. Data are representative of three (A) and two (C–G and I–K) independent experiments.

Article Snippet: Antibodies against CAMLG, NFAT2, NFAT4, HA-probe, and anti-Actin were from Santa Cruz Biotechnology, Inc. Anti-Flag, PMA, and calcium ionophore (A23187) were obtained from Sigma-Aldrich.

Techniques: Activation Assay, Infection, Stable Transfection, Expressing, Staining, Plasmid Preparation, Southern Blot, Quantitative RT-PCR, Luciferase, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

doi: 10.1084/jem.20131272

Figure Lengend Snippet: CsA treatment inhibits virulence by the local infection of WT, but not GRA6 -deficient, parasites. (A and B) MEFs stably expressing RFP-NFAT4 were infected with WT RH T. gondii (moi = 5) in the presence or absence of 100 ng/ml CsA or DMSO, fixed at 6 h after infection, and stained with DAPI. Bars, 5 µm. The representative images are shown in A and mean nuclear RFP signal was measured in B. The y axis represents the mean nuclear RFP signal (NFAT4) intensity over all the cells. (C) BALB/c mice ( n = 4 per each group) treated with 30 mg/kg CsA or the vehicle alone (control) were infected into right footpads with 10 3 WT luciferase-expressing parasites, and the progress of infection was assessed by BLI at indicated days after infection. The color scales indicate photon emission during a 60-s exposure. (D) Total photon emission analysis from mice ( n = 4 per each group) in C. Abdominal photon emission was assessed during a 60-s exposure. (E) BALB/c mice treated with CsA ( n = 9) or the vehicle alone ( n = 10) were infected into right footpads with 10 3 WT parasites, and the survival rates monitored for 20 d. P < 0.001 (CsA-treated vs. control, log-rank test). (F and I) Control or CsA-treated BALB/c mice ( n = 4 per each group) were intraperitoneally infected with 10 3 WT luciferase-expressing parasites (F), or infected into right footpads with 10 3 gra6-KO luciferase-expressing parasites (I), and the progress of infection was assessed by BLI at 7 d (F) or 11 d (I) after infection. The color scales indicate photon emission during a 60-s exposure. (G and J) Total photon emission analysis from control or CsA-treated BALB/c mice ( n = 4 per each group) in F or I. Abdominal photon emission was assessed during a 60-s exposure. N.S., nonsignificant. (H and K) Survival rates ( n = 10 per each group) were monitored for indicated days for mice in F or I. *, P < 0.001; **, P < 0.05; N.S., nonsignificant, determined by unpaired Student’s t test in B, D, G, or J. The red bars show means of the four samples (D, G, and J). Data are representative of two independent experiments (A–G, I, and J) or cumulative percentages of two independent (E, H, and K) experiments.

Article Snippet: Antibodies against CAMLG, NFAT2, NFAT4, HA-probe, and anti-Actin were from Santa Cruz Biotechnology, Inc. Anti-Flag, PMA, and calcium ionophore (A23187) were obtained from Sigma-Aldrich.

Techniques: Infection, Stable Transfection, Expressing, Staining, Control, Luciferase

Journal: The Journal of Experimental Medicine

Article Title: Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

doi: 10.1084/jem.20131272

Figure Lengend Snippet: NFAT4 is required for full virulence by WT parasites. (A) Schematic of the Cas9/gRNA-targeting site in the second of Nfat4 gene. Top: Structure of the Nfat4 gene. Black and white boxes denote the coding and noncoding exons, respectively. Middle: The target sequence for gRNA (underlined) and the PAM sequence were labeled with green and red, respectively. Bottom: The sequence of the mutated allele contained an insertion of 4 bp (dashed underlined and labeled with brown), resulting in a frameshift mutation. (B) Lysates of tail fibroblasts from indicated mice were subjected to Western blot with indicated Abs. (C) Quantitative RT-PCR analysis was performed using cDNA reversely transcribed from RNA extracted from WT parasite-infected (moi = 5) tail fibroblasts derived from WT or NFAT4 -deficient mice. Relative mRNA levels of indicated genes compared with the GAPDH level were shown in the y axis. Indicated values represent means ± SD of triplicates. (D) Representative flow cytometric plots of single cell suspension from the footpad of WT or NFAT4 -deficient mice at day 5 after infection of WT parasites was stained with 7-AAD and CD45.2. Then CD45 + and 7-AAD − populations were analyzed with Ly6G and CD11b. The percentages of CD11b + Ly6G + cells were shown. (E and F) Frequencies (E) and total numbers (F) of CD11b + Ly6G + cells of WT ( n = 4) or NFAT4 -deficient ( n = 3) mice infected with WT parasites at day 5 after infection. Indicated values represent means ± SD of all samples of each group. (G) WT or NFAT4 -deficient mice ( n = 4 per each group) were infected into right footpads with 10 3 WT luciferase-expressing parasites, and the progress of infection was assessed by BLI at indicated days after infection. The color scales indicate photon emission during a 60-s exposure. (H) Total photon emission analysis from WT or NFAT4 -deficient mice ( n = 4 per each group) in G. Abdominal photon emission was assessed during a 60-s exposure. The red bars show means of the four samples. (I) Survival rates ( n = 6 per each group) were monitored for 15 d for mice in G. P < 0.002 (WT vs. NFAT4 -deficient mice, log-rank test). *, P < 0.001; **, P < 0.05; ***, P < 0.01; N.S., nonsignificant, determined by unpaired Student’s t test in C, E, F, and H. Data are representative of two (B and D–H) or three (C) independent experiments or cumulative percentages of two independent (I) experiments.

Article Snippet: Antibodies against CAMLG, NFAT2, NFAT4, HA-probe, and anti-Actin were from Santa Cruz Biotechnology, Inc. Anti-Flag, PMA, and calcium ionophore (A23187) were obtained from Sigma-Aldrich.

Techniques: Sequencing, Labeling, Mutagenesis, Western Blot, Quantitative RT-PCR, Infection, Derivative Assay, Suspension, Staining, Luciferase, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

doi: 10.1084/jem.20131272

Figure Lengend Snippet: GRA6-mediated NFAT4 activation is strain-dependent. (A) The predicted amino acid sequence for the primary translation product of the GRA6 gene is shown for type I parasites on the top line. The sequences for type II and type III parasites are shown on the second and third line with a dash to indicate identity to the all strains sequence, respectively. (B) Lysates of 293T cells transfected with indicated expression vectors were subjected to Western blotting. (C) 293T cells were transfected with the NFAT-luc plasmid together with the indicated GRA6 expression vectors. Luciferase activities were expressed as indicated above. Error bars represent means ± SD of triplicates. (D and E) MEFs stably expressing RFP-NFAT4 were uninfected or infected with type I or type II T. gondii (moi = 5), fixed at 6 h after infection, and stained with DAPI. Bars, 5 µm. The representative images are shown in D and mean nuclear RFP signal was measured in E. The y axis represents the mean nuclear RFP signal (NFAT4) intensity over all the cells. *, P < 0.03, determined by unpaired Student’s t test in E. Data are representative of three (B and C) and two (D and E) independent experiments.

Article Snippet: Antibodies against CAMLG, NFAT2, NFAT4, HA-probe, and anti-Actin were from Santa Cruz Biotechnology, Inc. Anti-Flag, PMA, and calcium ionophore (A23187) were obtained from Sigma-Aldrich.

Techniques: Activation Assay, Sequencing, Transfection, Expressing, Western Blot, Plasmid Preparation, Luciferase, Stable Transfection, Infection, Staining

Journal: The Journal of Experimental Medicine

Article Title: Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

doi: 10.1084/jem.20131272

Figure Lengend Snippet: Polymorphisms on the C terminus determine the strain-specific NFAT4 activation and local infection-induced virulence. (A and B) 293T cells were transfected with the NFAT-luc plasmid, together with the indicated GRA6 expression vectors. Luciferase activities were expressed as indicated above. Error bars represent means ± SD of triplicates. A list of the point mutants of GRA6 expression vectors is shown (A and B, top). Type I, red; Type II, blue. (C) Lysates of 293T cells transiently cotransfected with Flag-tagged type I GRA6 variants and/or HA-tagged CAMLG were immunoprecipitated with the indicated Abs and subjected to Western blotting. Short and long indicate the time of exposure. (D) Lysates of indicated parasites were subjected to Western blotting using indicated Abs. (E) BALB/c mice ( n = 4 per each group) were infected into right footpads with 10 3 gra6-KO (parental line), KO+GRA6 WT, or KO+GRA6 V227E luciferase-expressing parasites, and the progress of infection was assessed by BLI at indicated days after infection. The color scales indicate photon emission during a 60-s exposure. (F) Total photon emission analysis from BALB/c mice ( n = 4) in E at indicated days after infection. Abdominal photon emission was assessed during a 60-s exposure. The red bars show means of the four samples. (G) The survival rates for mice in E ( n = 8 per each group) were monitored for 18 d. P < 0.001 (significant; KO+GRA6 WT vs. KO+GRA6 V227E, log-rank test). P > 0.05 (nonsignificant; gra6-KO vs. KO+GRA6 V227E, log-rank test). (H) 293T cells were transfected with the NFAT-luc plasmid together with the indicated GRA6 expression vectors. Luciferase activities were expressed as indicated above. Error bars represent means ± SD of triplicates. A list of the point mutants of GRA6 expression vectors is shown (H, left). *, P < 0.03; **, P < 0.01; ***, P < 0.001, determined by unpaired Student’s t test in F or H. Data are representative of three (A, B, and H) or two (C–F) independent experiments or cumulative percentages of two independent experiments (G).

Article Snippet: Antibodies against CAMLG, NFAT2, NFAT4, HA-probe, and anti-Actin were from Santa Cruz Biotechnology, Inc. Anti-Flag, PMA, and calcium ionophore (A23187) were obtained from Sigma-Aldrich.

Techniques: Activation Assay, Infection, Transfection, Plasmid Preparation, Expressing, Luciferase, Immunoprecipitation, Western Blot