Figure 2
Figure 2. T plastin expression is activated by calcium entry and down-regulated by calcineurin inhibitors and NFAT siRNA. (A) PBLs from patients with SS (n = 40), MF (n = 8), various hemopathies or undetermined erythroderma (others, n = 8), and from healthy donors (healthy, n = 16) were incubated with PMA and ionomycin combination (dark columns), ionomycin (gray columns), PMA (white columns), for 16 hours. For PBLs from patients with SS, 2 sets were distinguished, according to expression of constitutive T-plastin mRNA (SS PLS3+, n = 24) or not (SS PLS3−, n = 16). T-plastin mRNA expression was quantified by qRT-PCR. Results are presented as fold increase of T-plastin mRNA levels detected in stimulated cells relative to basal T-plastin mRNA levels detected in respective unstimulated cells. *P < .05, **P < .01, comparison of T-plastin mRNA levels from stimulated cells to T-plastin mRNA levels from respective nonstimulated cells. (B) Top panel: PBLs from patients with SS (n = 12) that constitutively expressed T-plastin mRNA were treated with EGTA (200μM) or FK-506 (10μM) or not treated (basal) for 16 hours. T-plastin mRNA expression was quantified by qRT-PCR. The constitutive T-plastin mRNA levels measured without treatment was reported to 100 to allow comparison between PBLs from SS patients. Results are presented as mean percentages (± SEM) of T-plastin mRNA levels obtained in the presence of EGTA (light gray bar) or FK506 (dark gray bar) relative to respective T-plastin levels detected in the absence of treatment (white bar). *P < .05, **P < .01, comparison of T-plastin mRNA levels from treated cells to T-plastin mRNA levels from basal nontreated cells. Bottom panel: PBLs from SS patients were not incubated (basal) or incubated with EGTA (200μM) or FK506 (10μM) for 16 hours. T-plastin protein expression was determined by immunoblot analysis with anti–T-plastin antibody and with GAPDH antibody to confirm equal loading. Results are representative of 3 independent experiments. (C) Top panel: HuT-78 cells constitutively expressed T-plastin mRNA. In a set of experiments, cells were preincubated with FK506 (10μM), or not for 16 hours before mRNA extraction. In another set of experiments, HuT-78 cells were transiently transfected with control siRNA (200 or 400nM), siRNA for NFAT1 (200nM), and NFAT2 (400nM) 24 hours before mRNA extraction. T-plastin mRNA expression was quantified by qRT-PCR and results are expressed in relative values of T-plastin expression of treated cells (gray) to respective basal nontreated cells (white) reported as 100 to allow experiment comparison (mean ± SEM, n = 6). **P < .01, comparison of T-plastin mRNA levels from treated cells to T-plastin mRNA levels from respective basal nontreated cells. Bottom panel: HuT-78 cells were transiently transfected with control siRNA (400nM), NFAT1 siRNA (200nM), or NFAT2 siRNA (400nM) 24 hours before total protein extraction was performed. NFAT1 and NFAT2 protein expression was determined by immunoblot analysis with anti-NFAT1 and anti-NFAT2 antibodies and with GAPDH antibody to confirm equal loading. Results are representative of 3 independent experiments. (D) Top panel: SeAx cells (0.2 × 106 cells/mL) were incubated (◊) or not (*) with PMA and ionomycin combination (PMA+Iono) over a 24-hour period (as triplicates) and collected for mRNA extraction at indicated times. T-plastin mRNA expression was quantified by qRT-PCR. Results are expressed in AUs and presented as mean ± SD (n = 3). In the absence of stimulation, SeAx cells did not express T-plastin mRNA (ΔCt values are less than 0.01 AU) whatever the collection time. Bottom panel: Cells were preincubated with FK-506 (10μM) for 1 hour before addition of PMA and ionomycin (PMA+Iono) for further 16 hours. In some experiments, SeAx cells were transiently transfected with control siRNA (200nM) or NFAT1 siRNA (200nM). Eight hours after, transfected cells were stimulated by addition of PMA and ionomycin (PMA+Iono) for 16 hours before mRNA extraction. T-plastin mRNA expression was quantified by qRT-PCR and results are expressed in arbitrary units (AU) and presented as mean ± SEM (n = 6). **P < .01, ***P < .001, comparison of T-plastin mRNA levels from treated cells to T-plastin mRNA levels from respective control cells.

T plastin expression is activated by calcium entry and down-regulated by calcineurin inhibitors and NFAT siRNA. (A) PBLs from patients with SS (n = 40), MF (n = 8), various hemopathies or undetermined erythroderma (others, n = 8), and from healthy donors (healthy, n = 16) were incubated with PMA and ionomycin combination (dark columns), ionomycin (gray columns), PMA (white columns), for 16 hours. For PBLs from patients with SS, 2 sets were distinguished, according to expression of constitutive T-plastin mRNA (SS PLS3+, n = 24) or not (SS PLS3−, n = 16). T-plastin mRNA expression was quantified by qRT-PCR. Results are presented as fold increase of T-plastin mRNA levels detected in stimulated cells relative to basal T-plastin mRNA levels detected in respective unstimulated cells. *P < .05, **P < .01, comparison of T-plastin mRNA levels from stimulated cells to T-plastin mRNA levels from respective nonstimulated cells. (B) Top panel: PBLs from patients with SS (n = 12) that constitutively expressed T-plastin mRNA were treated with EGTA (200μM) or FK-506 (10μM) or not treated (basal) for 16 hours. T-plastin mRNA expression was quantified by qRT-PCR. The constitutive T-plastin mRNA levels measured without treatment was reported to 100 to allow comparison between PBLs from SS patients. Results are presented as mean percentages (± SEM) of T-plastin mRNA levels obtained in the presence of EGTA (light gray bar) or FK506 (dark gray bar) relative to respective T-plastin levels detected in the absence of treatment (white bar). *P < .05, **P < .01, comparison of T-plastin mRNA levels from treated cells to T-plastin mRNA levels from basal nontreated cells. Bottom panel: PBLs from SS patients were not incubated (basal) or incubated with EGTA (200μM) or FK506 (10μM) for 16 hours. T-plastin protein expression was determined by immunoblot analysis with anti–T-plastin antibody and with GAPDH antibody to confirm equal loading. Results are representative of 3 independent experiments. (C) Top panel: HuT-78 cells constitutively expressed T-plastin mRNA. In a set of experiments, cells were preincubated with FK506 (10μM), or not for 16 hours before mRNA extraction. In another set of experiments, HuT-78 cells were transiently transfected with control siRNA (200 or 400nM), siRNA for NFAT1 (200nM), and NFAT2 (400nM) 24 hours before mRNA extraction. T-plastin mRNA expression was quantified by qRT-PCR and results are expressed in relative values of T-plastin expression of treated cells (gray) to respective basal nontreated cells (white) reported as 100 to allow experiment comparison (mean ± SEM, n = 6). **P < .01, comparison of T-plastin mRNA levels from treated cells to T-plastin mRNA levels from respective basal nontreated cells. Bottom panel: HuT-78 cells were transiently transfected with control siRNA (400nM), NFAT1 siRNA (200nM), or NFAT2 siRNA (400nM) 24 hours before total protein extraction was performed. NFAT1 and NFAT2 protein expression was determined by immunoblot analysis with anti-NFAT1 and anti-NFAT2 antibodies and with GAPDH antibody to confirm equal loading. Results are representative of 3 independent experiments. (D) Top panel: SeAx cells (0.2 × 106 cells/mL) were incubated (◊) or not (*) with PMA and ionomycin combination (PMA+Iono) over a 24-hour period (as triplicates) and collected for mRNA extraction at indicated times. T-plastin mRNA expression was quantified by qRT-PCR. Results are expressed in AUs and presented as mean ± SD (n = 3). In the absence of stimulation, SeAx cells did not express T-plastin mRNA (ΔCt values are less than 0.01 AU) whatever the collection time. Bottom panel: Cells were preincubated with FK-506 (10μM) for 1 hour before addition of PMA and ionomycin (PMA+Iono) for further 16 hours. In some experiments, SeAx cells were transiently transfected with control siRNA (200nM) or NFAT1 siRNA (200nM). Eight hours after, transfected cells were stimulated by addition of PMA and ionomycin (PMA+Iono) for 16 hours before mRNA extraction. T-plastin mRNA expression was quantified by qRT-PCR and results are expressed in arbitrary units (AU) and presented as mean ± SEM (n = 6). **P < .01, ***P < .001, comparison of T-plastin mRNA levels from treated cells to T-plastin mRNA levels from respective control cells.

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