Figure 8
From: ITF2357 transactivates Id3 and regulate TGFβ/BMP7 signaling pathways to attenuate corneal fibrosis
ITF2357 attenuates anti-fibrotic functions through Id3 regulation.
(A–D) All Id proteins (Id1-4) transcripts were expressed in pHCSFs. ITF2357 alone induced a significant increase in the expression of (A) Id1, (B) Id2, (C) Id3 and (D) Id4. (C) Id3 mRNA showed a striking 10-fold increase in the presence of TGFβ + ITF2357. (E) Immunostaining of Id3 protein in pHCSFs showed decreased expression in TGFβ only treated group, but an increased expression in the ITF2357 treated groups regardless of TGFβ. Scale bar = 50 μm. (F–K) Subsequent Id3 knockdown using Lipofectamine transfection of Id3 siRNA showed up-regulation of (F) αSMA in Id3 siRNA + TGFβ + ITF2357 group to the level of that seen in TGFβ treated group only. (G) BMP7 mRNA production in Id3 knockdown remained unchanged. (H) Id1, (I) Id2 and (K) Id4 mRNA was upregulated in Id3 knockdown condition suggesting a compensatory mechanism. (J) Id3 siRNA transfection efficiency was found to be ~75%. *p < 0.05; **p < 0.01 and ***p < 0.001 against control. $$$p < 0.001 against TGFβ + ITF2357. There were n = 6–10 in mRNA expression and n = 4 in immunostaining for each group. Error bars represent SEM.
