Table 1.
Yeast strains used in this study.
Fig 1.
Clausmarin A alleviates the Ca2+-dependent growth inhibition of the YNS17 (Δzds1) yeast strain.
YNS17 cells (5 x 106 cells/mL) were incubated for 30 min in YPD medium containing the indicated concentration of clausmarin A before the addition of CaCl2 to 100 mM final concentration and incubating at 30°C with shaking for the indicated time. Legend: 100 mM CaCl2 (■);100 mM CaCl2 + 125 μM clausmarin A (□);100 mM CaCl2 + 250 μM clausmarin A (▲);100 mM CaCl2 + 250 nM FK506 (△); none (○) and 250 μM clausmarin A (●). Data shown are from one trial and are representative of those seen from three independent trials. *Statistically different with p-value <0.0001.
Fig 2.
Clausmarin A inhibits the calcineurin but not the Mpk1 pathway.
The effect of clausmarin A on the growth of the loss-of-function mutations in the (A) calcineurin (Δcnb1) or (B) the Mpk1 MAP kinase (Δmpk1) Ca2+-signaling pathways. The experimental procedures were similar to that described in the legend to Fig 1, except no CaCl2 was added. Legend: no addition (○); 250 μM clausmarin A (▲); 250 nM FK506 (△). Data shown are from one trial and are representative of those seen from three independent trials. *Statistically different with p-value <0.0001.
Fig 3.
Clausmarin A alleviates the growth inhibition of a constitutively active calcineurin.
(A) The effect of clausmarin A on the YRC1 yeast strain expressing an inducible constitutively active form of calcineurin. The experimental procedures were similar to those described in the legend to Fig 1, except the YRC1 strain with a chromosomally integrated construct for the galactose-inducible constitutively active form of the calcineurin catalytic subunit (CMP2ΔC) and the appropriate medium (SC) without CaCl2 addition for induction of GAL1 promoter was used. Legend: SC medium with no addition (□); 250 μM clausmarin A (▲); 250 nM FK506 (△). The GAL1 promoter was induced by the addition of 2% (w/v) galactose and incubated at 30°C with shaking for the indicated period of time. *Statistically different with p-value <0.001. (B) The samples obtained after 12 h of incubation were observed under phase-contrast microscopy (left), fluorescence microscopy of Hoechst 33342-stained cells (middle) or flow cytometric analysis of Hoechst 33342-stained cells (right). Data shown are from one trial and are representative of those seen from three independent trials.
Fig 4.
Clausmarin A inhibits IL-2 and IL-2 mRNA production in Jurkat cells.
(A) Jurkat cells were treated with various concentrations of clausmarin A or with 100 nM FK506 for 30 min, then stimulated with 25 ng/mL PMA and 1 μg/mL Io at 37°C for 24 h. The treatments were: (1) none, (2) 0.5% (v/v) DMSO, (3–7) clausmarin A at (3) 0.25 μM, (4) 1 μM, (5) 2.5 μM, (6) 5 μM and (7) 25 μM, (8) 100 nM FK506 and (9) unstimulated (no PMA/Io treatment). (B) Effect of clausmarin A on IL-2 mRNA expression. Jurkat cells were treated with various concentrations of clausmarin A or with 100 nM FK506 for 30 min prior to stimulation with PMA/Io as described in (A). The various treatments were: (1) none, (2–4) clausmarin A at (2) 5 μM, (3) 25 μM and (4) 50 μM, (5) 100 nM FK506 and (6) unstimulated (no PMA/Io treatment). RNA was extracted and subjected to two-stage gene specific sqRT-PCR. (C) Corrected IL-2 expression levels relative to that of β-actin. Data are shown as the mean ± 1 SD, derived from three replicated. Means that are significantly different from the untreated cells at p ≤ 0.001 are indicated by *.
Fig 5.
Clausmarin A inhibits NFAT dephosphorylation in Jurkat cells.
Jurkat cells were treated with various concentrations of clausmarin A or FK506 for 30 min at 37°C and then activated with 25 ng/mL PMA and 1 μg/mL Io at 37°C for 24 h. The treatments were: (1) unstimulated, (2–8) PMA/Io stimulated following treatment with (2) none, (3–7) clausmarin A at (3) 12.5 μM, (4) 25 μM, (5) 50 μM, (6) 75 μM and (7) 100 μM, and (8) 100 nM FK506. (A) Western blot showing the dephosphorylated and phosphorylated forms of NFAT and that of the β-actin loading control. Data are representative of three replicate experiments (B). The relative amount of dephosphorylated NFAT to the β-actin loading control. Data are shown as the mean ± 1 SD, derived from three replicated. Means that are significantly different from the untreated cells at p ≤ 0.001 are indicated by *.