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1.
Figure 2

Figure 2. From: Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection.

Cytokine and chemokine induction by T. gondii infection. Human IL-8, CCL15, CCL20, and CCL24 gene transcripts levels were measured by Real-time PCR analysis 4 hours post T. gondii infection. The data are normalized to GAPDH and compared against uninfected expression levels (mRNA REL %, relative expression levels).

Chia-Hsin Ju, et al. J Immunol. ;183(11):7420-7427.
2.
Figure 5

Figure 5. From: Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection.

TLR genes are expressed in Henle 407 cells. (A) Total RNA was collected from Henle 407 cells, reverse transcribed to cDNA and then amplified for human TLR or GAPDH by PCR. Genomic DNA was used as a positive control. (B) Henle 407 cells were treated with 10 ng/mL PMA as positive control or TLR1/2, TLR4, TLR2/6 and TLR9 ligands (1 μg/mL Pam3Cys, 100 ng/mL LPS, 1 μg/mL Malp-2, and 5 μg/mL CpG respectively). Total cell lysate were collected and immunoblotted for total and phosphorylated forms of ERK1/2 and p38.

Chia-Hsin Ju, et al. J Immunol. ;183(11):7420-7427.
3.
Figure 4

Figure 4. From: Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection.

T. gondii induced MAPK activation and IL-8 secretion is MyD88 dependent. (A) Henle 407 cells were either untransfected, transfected with control shRNA plasmids, or MyD88 shRNA plasmids carrying the MyD88 RNA interference sequence. Cell lysates from the stably transfected cell lines were collected for immunoblotting with antibodies against MyD88 or β-tubulin. (B) Activation of ERK1/2 in T. gondii infected MyD88 knockout Henle 407 cells. Control Henle 407 cells, control shRNA cells, and MyD88 shRNA cells were infected with RH tachyzoites (parasite to cell ratio 6:1) at the indicated time points. Cell lysates were collected for immunoblotting with antibodies against total and phosphorylated forms of ERK1/2. (C) Control Henle 407, control shRNA, and MyD88 shRNA cells were infected with T. gondii (parasite to cell 6:1). Supernatants from each sample were collected for IL-8 ELISA analysis. (Triplicate assays, error bar = SD).

Chia-Hsin Ju, et al. J Immunol. ;183(11):7420-7427.
4.
Figure 7

Figure 7. From: Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection.

MAPK activation and TLR2 dependent response to T. gondii is not strain type specific. (A) Henle 407 cells were infected with, GT-1 (Type I), CC, DEG (Type II), and VEG (Type III) T. gondii strains for the indicated time points. Total cell lysates were collected for immunoblotting with antibodies against total and phosphorylated forms of ERK1/2 and p38. (B) HEK293 cells were transfected with human TLR2 and an NF-κB luciferase reporter plasmid. The transfected HEK293 cells were stimulated with TLR2 ligand Pam3Cys or different T. gondii tachyzoite strains. Cell lysates were collected at 18 hr and assayed for luciferase activity. (Triplicate assays, error bar = SD).

Chia-Hsin Ju, et al. J Immunol. ;183(11):7420-7427.
5.
Figure 1

Figure 1. From: Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection.

Intestinal epithelial cells respond to T. gondii infection. (A) Henle 407 cells were infected with RH tachyzoites (parasite to cell ratio 6:1) for the indicated times. Whole cell lysates were collected for immunoblotting with antibodies against total and phosphorylated forms of ERK1/2 and p38 MAPK. (B) IL-8 levels in infected culture supernatants were collected at the indicated time points and assayed by ELISA (triplicate assays, error bar = SD). Experiments were performed 3 times with similar results. *, p < 0.05 compared with IL-8 levels of uninfected cells. (C and D) Henle 407 cells were infected with RH (C) or RH-YFP (D) parasites for the indicated times. NF-κB p65 localization was visualized by immunoblotting of cytosolic and nuclear extracts (C) or by immunofluorescent staining (D) for anti–NF-κB p65 (red).

Chia-Hsin Ju, et al. J Immunol. ;183(11):7420-7427.
6.
Figure 3

Figure 3. From: Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection.

T. gondii induced MAPK activation and IL-8 secretion in intestinal epithelial cells is PI-3 kinase signaling independent. (A) Henle 407 cells were preincubated 2 hr with or without PI-3 kinase inhibitor wortmannin (WM, 50 ng/mL) followed by infection with T. gondii (parasite to cell ratio 6:1). At the indicated time points, cell lysates were collected for immunoblotting Akt and ERK1/2. The blot was reprobed for total ERK1/2. (B) Henle 407 cells were pretreated with or without wortmannin and infected with T. gondii as described above. IL-8 levels in infected culture supernatants were collected at the indicated time points and assayed by ELISA (triplicate assays, error bar = SD). *, p < 0.05 compared with IL-8 levels of uninfected cells. (C) Henle 407 cells were pretreated with or without wortmannin and infected with T. gondii as described above. Analysis of IL-8 and GAPDH transcripts was performed by Real-time PCR. Each sample was normalized to internal GAPDH expression levels (IL-8 mRNA REL %, relative expression levels).

Chia-Hsin Ju, et al. J Immunol. ;183(11):7420-7427.
7.
Figure 6

Figure 6. From: Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection.

Human TLR2 is involved in recognition of T. gondii. (A) HEK293 cells were transfected with different human TLRs and an NF-κB luciferase reporter plasmid. The transfected HEK293 cells were stimulated with TLR ligands, STAg, or infected with live RH tachyzoites. Cell lysates were collected and assayed for luciferase activity (Triplicate assays, error bar = SD). (B-D) Henle 407 cells were transiently transfected with shRNA plasmids carrying TLR2 (2), MyD88 (M) RNA interfering sequences, or no DNA control (C). (B) TLR2 and MyD88 mRNA levels as measured by Real-time PCR. (C) Total cell lysates from cells infected with RH tachyzoites (parasite to cell ratio 6:1) for the indicated time points were immunoblotted with antibodies against total and phosphorylated forms of ERK1/2 and p38. (D) IL-8 levels from cells in B as measured by Real-time PCR following 4 hour infection with T. gondii RH tachyzoites. (E) HEK293 cells were transfected with TLR2 and an IL-8 promoter-luciferase reporter construct. Luciferase activity from cells left in media alone (control), or infected with tachyzoites (RH strain). Cells were pretreated with ERK1/2 inhibitor U0126, or DMSO as a solvent control, prior to infection. (Triplicate assays, error bar = SD)

Chia-Hsin Ju, et al. J Immunol. ;183(11):7420-7427.

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