Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Immunostaining of T84 cell monolayers for (A) occludin and (B) ZO‐1 showing the formation of a continuous monolayer of cells. (C, D) mRNA levels of CYP1A1 and CYP1B1 cultured in 24‐well plates (‘24w’) and as monolayers on Transwell inserts. The individual ΔCt values (the difference in threshold cycle between the mRNA of the CYP and the housekeeper mRNA [RPL19]) are shown with the means represented by the bars. The right Y‐axes show data where the mean for the 24‐well plates is set to 100%.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Immunostaining, Cell Culture

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effect of IFNγ treatment of T84 cells upon their oxylipin content (fmol per well)

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Control

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effects of IFNγ and TMS upon the mRNA expression of HIF1, AhR and proteins involved in the barrier function of T84 cells

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Expressing, Control

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Panel A, effects of vehicle (V, N = 21 and 20 for control and IFNγ‐treated respectively), and TMS (T, 100 nM; N = 13 and 19 for control and IFNγ‐treated respectively) treatment upon TEER levels in T84 cells cultured in Transwells. Vehicle or IFNγ (10 ng·mL−1) was added basolaterally and vehicle or TMS apically prior to culture for 24 h. In all cases the values shown are the TEER values at 24 h expressed as a percentage of the corresponding values for the same conditions at t = 0. As the sample sizes are different, we calculated ANOVA P values using a permutation test (function aovp in the package lmPerm for R). The P values for IFNγ, TMS and IFNγ × TMS were all <0.05. Pairwise P values (after controlling for α inflation using a Bonferroni correction) were calculated using two‐sided permutation tests using the function permTS in the package perm for R and are shown in the Figure: *P < 0.05; significantly different as indicated; NS, not significantly different. Panel B, % reversal of the effect of IFNγ upon TEER by TMS (N = 19), benzo[a]pyrene (Benzo, 10 μM, N = 6)) and α‐naphthoflavone (α‐Nap, 100 nM, N = 6). For each experiment, % reversal was calculated as [100 – (100 × (ΔTEERcpd‐con − ΔTEERcon)/(ΔTEERifn − ΔTEERcon)], where ‘ΔTEER’ refers to the change (in Ω·cm2 seen between t0 and t24 for the different conditions; ‘cpd’ refers to the combination of test compound and IFNγ treatment, ‘con’ to the untreated cells and ‘ifn’ to cells treated with IFNγ and vehicle. A one‐way permutation ANOVA (independence_test in the package coin for R) gave a P value <0.05. One sample t‐tests of each condition versus 0% gave a significant P value for TMS, but not for benzo[a]pyrene or α‐naphthoflavone.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Control, Cell Culture

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effects of vehicle (v) and TMS (T, 100 nM) upon the mRNA for A, IL8 and B, TNFα (N = 6, except for the TMS‐treated control cells, where N = 5) in control and IFNγ‐treated cells. The T84 cells in Transwells were treated as described in Figure 4. For the IFNγ‐treated cells, the mean values for vehicle‐ and TMS‐treated cells were not significantly different.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Control

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effect of IFNγ treatment upon the properties of T84 cells cultured in monolayers on Transwells. IFNγ (10 ng·mL−1) was added basolaterally at t0 and the cells were cultured for 24 h. Panel A, TEER values for the controls (N = 31) and IFNγ‐treated (N = 30) cells are pooled from several experimental series. In all the scatterplots presented in this paper, the solid bars show the mean values. A two‐way type 3 ANOVA with time as repeated measure gave F 1,59 values for time, IFNγ and the interaction time × IFNγ of 140, 10.0 and 235, respectively (all statistically significant); *P < 0.05; significantly different as indicated; Sidak's multiple comparisons test. Residual plots (of a corresponding linear mixed model analysis) indicated no obvious heteroscedasticity or non‐normality of the distribution. Panel B, mRNA levels for CYP1A1 (N = 10), CYP1B1 (N = 10), IL8 (N = 8) and TNFα (N = 8) following 24 h of treatment with either vehicle or IFNγ. *P < 0.05; significantly different as indicated; two‐tailed t‐tests.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Cell Culture, Two Tailed Test

Journal: British Journal of Pharmacology

Article Title: Involvement of CYP1B1 in interferon γ‐induced alterations of epithelial barrier integrity

doi: 10.1111/bph.14122

Figure Lengend Snippet: Effect of PDTC (100 μM) and YC‐1 (50 μM) upon mRNA expression of (A) CYP1A1 and (B) CYP1B1 for IFNγ‐treated T84 cells (N = 6). Cells were seeded overnight in 24‐well plates and incubated with vehicle or the compounds for 1 h, before incubation with IFNγ (10 ng·mL−1) for 8 h. *P < 0.05; significantly different from control (concomitantly cultured cells in the absence of IFNγ); two‐tailed paired t‐test. For the IFNγ‐treated cells, Levene's test for homogeneity of variance was significant for CYP1B1 and so, we analysed the data using a two‐way permutation test (function aovp in the package lmperm for R). For CYP1A1, a significant P value was found for the main effect of YC‐1. For CYP1B1, significant P values were found for YC‐1 and the interaction PDTC × YC‐1.

Article Snippet: Cell culture Human T84 colon carcinoma cells (American Type Culture Collection, Rockville, MD, USA) that had been selected for capacity to form polarized, tight monolayers on semipermeable support (Ou et al., 2009a ) and thereafter frozen directly after expansion were cultured at 37°C in a 5% CO 2 environment in DMEM/F12 medium supplemented with 8% FBS, 1% penicillin + streptomycin (PEST) and 1% L‐glutamine.

Techniques: Expressing, Incubation, Control, Cell Culture, Two Tailed Test

Journal: PLoS ONE

Article Title: Campylobacter jejuni Type VI Secretion System: Roles in Adaptation to Deoxycholic Acid, Host Cell Adherence, Invasion, and In Vivo Colonization

doi: 10.1371/journal.pone.0042842

Figure Lengend Snippet: (A) T84 adhesion efficacy, B) T84 invasion efficacy, (C) RAW 267.4 macrophage adhesion efficacy and (D) RAW 267.4 macrophage invasion efficacy. The graph represents results from three independent experiments. Error bars represent the standard error of the mean. P value: *≤0.05, **≤0.01, ***≤0.001.

Article Snippet: A stock culture of T84 (a human colonic epithelial cell line; ATCC, CCL 6) was maintained in 1∶1 mixture of Han's F12 and Dulbecco's modified Eagles medium (DMEM∶F12; ATCC) supplemented with 5% (vol/vol) fetal bovine serum, penicillin (100 U ml −1 ) and streptomycin (100 μg ml −1 ) according to ATCC's recommendation.

Techniques:

Journal: Bioactive Materials

Article Title: Engineered a dual-targeting HA-TPP/A nanoparticle for combination therapy against KRAS-TP53 co-mutation in gastrointestinal cancers

doi: 10.1016/j.bioactmat.2023.10.003

Figure Lengend Snippet: Abrogation of mutp53 protein conferred GOF phenotypes and co-targeting effects in vitro. A) Relative mRNA level of mutp53 downstream pathway in MIA PaCa-2 (p53 R248W ) cells treated with or without HA-TPP/A (5 μM) for 24 h, analyzed by RT-PCR. The values of the HA-TPP/A group were normalized against these of the control group. B) Cytotoxicity tests of the normal intestinal epithelial cells (HCoEpiC), normal hepatocytes (LO2), wtp53 CRC cell (T84), mutp53 cells MIA PaCa-2 (p53 R248W ) and SW837 (p53 R248W ) after the indicated treatment for 24 h by HA-TPP/A. Data are represented as means ± SD (n = 3). C) Cytotoxicity tests after 24 h by treatment of HA-TPP/A 10 μM (AMG510 100 nM; HA-TPP 10 μM) in HCoEpiC, LO2, T84, MIA PaCa-2 and SW837 cells. D) Cell viability on MIA PaCa-2 cells after the indicated treatment for 24 h by AMG510, HA-TPP, A + T (AMG510 plus HA-TPP) and HA-TPP/A. Data are represented as means ± SD (n = 3). E) Viability of MIA PaCa-2 cells after the indicated treatment for 24h. Dosing: AMG510 (100 nM), HA-TPP (10 μM), A + T (AMG510 100 nM plus HA-TPP 10 μM), HA-TPP/A (10 μM). F) Clonal formation assay of control MIA PaCa-2 and SW837 cells and after treatment with AMG510, HA-TPP, AMG510+HA-TPP, HA-TPP/A for 12 d at the indicated condition. Dosing: AMG510 (10 nM), HA-TPP (1 μM), A + T (AMG510 10 nM plus HA-TPP 1 μM), HA-TPP/A 1 μM (AMG510 10 nM; HA-TPP 1 μM). G) Cell migration inhibition of MIA PaCa-2 cells after the indicated treatment for 24 h. Dosing: AMG510 (100 nM), HA-TPP (10 μM), A + T (AMG510 100 nM plus HA-TPP 10 μM), HA-TPP/A (10 μM). Data are represented as means ± SD (n = 3). H) Flow cytometric analysis of cell apoptosis of MIA PaCa-2 cells after different treatments for 24 h. Dosing: AMG510 (100 nM), HA-TPP (10 μM), A + T (AMG510 100 nM plus HA-TPP 10 μM), HA-TPP/A (10 μM). (*p < 0.05 , ***p < 0.001 and ****p < 0.0001).

Article Snippet: Four human CRC cell lines (SW837, HCT8, T84, DLD-1), two human PDAC cell lines (MIA PaCa-2, PATU-8988S), human normal liver cell line (LO2) and human normal colon cell line (HCoEpic) used in this study were purchased from ATCC.

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Control, Tube Formation Assay, Migration, Inhibition