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Figure legends
Figure 1. Arecoline decreased membrane expression of ZO-1 in confluent TM4 cells.
These were treated with indicated arecoline concentration for 6 hours. (A) Morphology of confluent TM4 treated with (right panel) or without (left panel) 400 μM of arecoline were visualized. Treated cells (right panel) displayed flat and spread
morphology compared to controls (left panel). (B) ZO-1 protein expression detected by immunofluorescent assay was visualized by fluorescent microscope. Arecoline-treated cells were manifested lower ZO-1 signal than controls. (C) Insoluble and soluble fractions of cell lysates were prepared for Z0-1 detection by western blot. (D) Inhibitory effect of arecoline on insoluble ZO-1 is plotted on bar graph. Signals of insoluble ZO-1 were quantified by densitometry analysis and expressed as average percentage of respective control cells from three independent experiments to rate arecoline’s inhibitory effect (*P<0.05 versus 0 μM controls). (E) The amounts of ZO-1 mRNA were determined using RT-qPCR. Results depict mean±SD of three independent experiments. *P<0.05 compared with cells incubated without arecoline treatment (0 μM).
Figure 2. Arecoline activates ERK1/2 MAPKs and increases TNF-alpha production.
(A) Protein signal of phospho-ERK1/2, ERK1/2, phospho-JNK, phospho-IkappaB-alpha, phospho-PP2A, phospho-STAT1 and GAPDH in arecoline (400μM) treated cells were detected by western blot. Arecoline induces phosphorylation of ERK1/2 MAPKs. These same membranes were stripped and re-detected by antibodies of total ERK1/2 and GAPDH. Results shown represent three independent experiments. (B)(C) No obvious cell death in arecoline-treated TM4 cells treated with indicated arecoline
concentration, cytotoxicity or viability gauged by released LDH activity detection kit after 6 hours (B) or MTS Assay after 6, 12 and 24 hours of treatment (C). Data express percentage of control cells (*P<0.05 versus 0 μM control group). (D)(E) Arecoline induced TNF-alpha mRNA (D) and soluble protein production (E). Cells were treated with indicated concentration of arecoline for 6 hours, levels of TNF-alpha mRNA measured by RT-qPCR and soluble protein in culture medium measured by ELISA. Results depict mean±SD of three independent experiments. *P<0.05 compared with cells incubated without arecoline treatment (0 μM).
Figure 3. Activation of ERK1/2 MAPKs is important for TNF-alpha induction and ZO-1 protein redistribution by arecoline in TM4 cells. (A) PD98059 pretreatment inhibited ERK1/2 activation. Cells were stimulated with or without 400 μM of arecoline for 10 minutes after pretreatment with 10 µM PD98059 for 1 hour, levels of ERK1/2 and phospho-ERK1/2 determined by western blot. Results shown represent three independent experiments. (B) (C) PD98059 pretreatment inhibited arecoline-induced increase in mRNA (B) and soluble protein (C) of TNF-alpha. Cells were treated with or without arecoline for 6 hours after PD98059 pretreatment. Levels of mRNA and soluble protein of TNF-alpha were measured by RT-qPCR and ELISA respectively. Results depict mean±SD of three independent experiments. *P<0.05
compared with cells incubated without arecoline treatment (mock control). (D) PD98059 pretreatment rescued arecoline-induced protein redistribution of ZO-1.
Levels of insoluble or soluble ZO-1 were determinate by western blot. Results represent three independent experiments. (E) Effect of arecoline and PD98059 on insoluble ZO-1 is plotted on bar graph. Signals were quantified by densitometry analysis and expressed as average percentage of respective control cells from three independent experiments (*P<0.05 versus mock controls).
Figure 4. ERK1/2 MAPKs are involved in increases of mRNA and soluble protein TNF-alpha by arecoline in THP1 cells. (A) (B) Arecoline increased mRNA (A) and soluble protein (B) of TNF-alpha. THP1 (2×106 in 1.5 mL) cells were treated with indicated doses of arecoline for 6 hours. (C) (D) PD98059 inhibited TNF-alpha mRNA (C) and soluble protein (D) inductioin via arecoline. Cells were pretreated with 10 µM PD98059 for 1 h and further treated with 400 µM arecoline for 6 hours.
Those mRNA and soluble protein of TNF-alpha levels were measured by RT-qPCR and ELISA. These results represent mean±SD of three independent experiments;
asterisk shows significant difference (p < 0.05) compared with controls (0 μM).
Tables
Table 1: Data collection of significant fold-changes for detected proteins from Micro-Western Array analysis
Arecoline treated time
Antibodies 10 minutes 60 minutes
Increased Phospho-p44/42 MAPK (Thr202/Tyr204) 1.98 Phospho-p38 MAPK (Thr180/Tyr182) 1.51
Phospho-IkappaB-alpha (Ser36) 1.33
Phospho-Jun(-c) (Ser63) 1.6
Phospho-mTOR (Thr2446) 1.16 2.3
Decreased Phospho-PP2A (Tyr307) 0.89 0.68
Phospho-STAT1 (Ser727) 0.6
Phospho-Src (Tyr418) 0.85 0.79
Note: Relative protein abundance was normalized to the average of GAPDH and actin. A fold change of 1.3 or 0.7 in expression between 0 and 10 minutes or 0 and 60 minutes groups was used as a cutoff.
Figure 1
Figure 2
Figure 3
Figure 4