Fig 1.
Enarodustat inhibits FSL-1-induced TSLP expression.
(A-C) TSLP mRNA expression levels in HaCaT cells 2 h after TNF-α (A), PAR2-agonist + IL-4 (B), and FSL-1 (C) stimulation. Cells were treated with enarodustat 24 h before stimulation. mRNA expression was determined by RT-qPCR. (D) Concentration-dependent effect of enarodustat on FSL-1-induced TSLP mRNA expression. *p < 0.05, **p < 0.01 vs. FSL-1 alone group. Data are represented as the mean ± S.E.M. (n = 3). N.S., not significant.
Fig 2.
NF-κB activation is not affected by enarodustat treatment.
(A) Expression levels of IκBα after enarodustat and FSL-1 treatment. Samples were collected 30 min after FSL-1 stimulation. IκBα and α-tubulin proteins levels were determined by western blotting. α-tubulin was used as a loading control. (B) Expression NF-κB p65 in nuclear fraction. Histone H3 was used as a loading control for the nuclear fraction samples. α-tubulin was used as a loading control for the total cell lysate. (C) Concentration-dependent effects of TPCA-1 on TSLP mRNA expression in FSL-1-stimulated HaCaT cells. (D) Effects of enarodustat on TSLP mRNA expression after TPCA-1 treatment. *p < 0.05, **p < 0.01 vs. FSL-1 alone group. Data are represented as the mean ± S.E.M. (n = 3). N.S., not significant.
Fig 3.
Enarodustat regulates AP-1 activation by suppressing JNK phosphorylation.
(A) Concentration-dependent effects of T-5224 on TSLP mRNA expression in FSL-1-stimulated HaCaT cells. (B) Effects of enarodustat on TSLP mRNA expression after T-5224 treated. (C) Expression levels of phospho-ERK, p38 and JNK after enarodustat and FSL-1 treatment. Samples were collected 30 min after FSL-1 stimulation. Phospho-MAPKs and GAPDH proteins were determined by western blotting. GAPDH was used as a loading control. (D) Intensity ratio of phospho-JNK/GAPDH. Band intensity was quantified using the ImageJ software. *p < 0.05, **p < 0.01 vs. FSL-1 alone group. Data are represented as the mean ± S.E.M. (n = 3). N.S.; not significant.
Fig 4.
Cobalt chloride suppresses FSL-1-induced TSLP expression and JNK phosphorylation.
(A) Effects of CoCl2 on FSL-1 induced TSLP mRNA expression. CoCl2 (0.4 mM) was treated 24 h before FSL-1 stimulation in HaCaT cells. (B) Expression levels of HIF1α, phosphor-JNK and GAPDH. Samples were collected 30 min after FSL-1 stimulation. GAPDH was used as a loading control. (C) Intensity ratio of phospho-JNK/GAPDH. Band intensity was quantified using the ImageJ software. *p < 0.05, **p < 0.01 vs. FSL-1 alone group. Data are represented as the mean ± S.E.M. (n = 3).
Fig 5.
JNK inhibitor suppresses FSL-1-induced TSLP expression.
(A) Expression levels of phospho-JNK after SP600125 and FSL-1 treatment. Samples were collected 30 min after FSL-1 stimulation. Phospho-JNK and α-tubulin proteins were determined by western blotting. α-tubulin was used as a loading control. (B) Effects of SP600125 on FSL-1 induced TSLP mRNA expression. (C and D) Effects of T-5224 on TSLP mRNA expression levels induced by TNF-α (C) or PAR2-agonist + IL-4 (D). (E) The effects of SP600125 on TSLP mRNA expression induced by PAR2-agonist + IL-4. *p < 0.05, **p < 0.01 vs. stimulation alone group. Data are represented as the mean ± S.E.M. (n = 3-4). N.S.; not significant.
Fig 6.
Enarodustat induces JNK-related DUSP family expression.
DUSP mRNA expression 24 h after enarodustat (10 μM) treatment was determined by RT-qPCR. mRNA expression in the enarodustat-treated group was normalized to that in the untreated control group. Normalized expression was indicated as a fold change in mRNA expression. *p < 0.05, **p < 0.01 vs. untreated control group. Data are represented as the mean ± S.E.M. (n = 3).
Fig 7.
Knockdown of HIF attenuates the suppression of TSLP expression by enarodustat.
HaCaT cells were transfected with siRNAs at the time of seeding. Cells were treated with enarodustat 24 h before FSL-1 stimulation. (A and B) mRNA expression levels of HIF1α (A) and HIF2α (B) 72 h after siRNA transfection. *p < 0.05, **p < 0.01 vs. control siRNA-treated group. Data are represented as the mean ± S.E.M. (n = 3). (C) Protein expression levels of HIF1α in enarodustat- and FSL-1-treated groups after control or HIF siRNA treatment. (D) Intensity ratio of HIF1α/α-tubulin in enarodustat treated groups under control siRNA or HIF siRNA treated condition. Band intensity was quantified using the ImageJ software. (E) TSLP mRNA expression levels in control- and HIF siRNA-treated cells. *p < 0.05, **p < 0.01 vs. FSL-1 alone group. Data are represented as the mean ± S.E.M. (n = 3). N.S.; not significant.
Fig 8.
Knockdown of HIF attenuates JNK dephosphorylation by enarodustat.
(A) Fold change in DUSP1, 3, 4, 6, 9 mRNA expression levels 24 h after enarodustat treatment in the control- (black column) or HIF siRNA- (white column) treated group. mRNA expression in the enarodustat-treated groups was normalized to that in the untreated control group. Normalized expression was indicated as fold change in mRNA expression.. (B) Protein expression levels of phospho-JNK protein in enarodustat- and FSL-1-treated groups after control or HIF siRNA transfection. (C) Intensity ratio of phospho-JNK/GAPDH in FSL-1 alone groups and FSL-1 + enarodustat treated groups was quantified using the ImageJ software. JNK phosphorylation ratio was calculated to devide the intentisy ratio of FSL-1 + enarodustat group by that of FSL-1 alone group. *p < 0.05, **p < 0.01 vs. corresponding control siRNA-treated group. Data are represented as the mean ± S.E.M. (n = 3).