XB-IMG-139013
Xenbase Image ID: 139013
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FIGURE 6. Hspa5 is required for transduction ofRAsignaling. A,Hspa5MO2down-regulates lhx1, cdx4, and gbx2 gene expression in animal caps treated with
atRA but not in animal caps treated with activin. Hspa5MO2 (MO2) (30 ng/embryo) was injected into both blastomeres of Xenopus embryos at the two-cell
stage. Animal caps were dissected at stage 9, treated with either atRA or activin separately or a mixture of both for 3 h, and then cultured in normal animal cap
culture medium for another 3 days. Expression of the indicated genes was examined by RT-PCR. Depletion of Hspa5 attenuates expression of lhx1, cdx4, and
gbx2 induced by atRA. Activin treatment up-regulates lhx1, hoxd1, cdx4, gbx2, and chordin expression. Knockdown of Hspa5 has little effect on the indicated
genes except that lhx1 expression is enhanced. Treatment of atRA and activin induces lhx1, hoxd1, and gbx2 expression in control animal caps, whereas
knockdown of Hspa5 causes a reduction of lhx1 but not hoxd1 or gbx2. AC, control animal cap; WE, sibling whole embryos. B, Hspa5MO decreases expression
of atRA-responsive genes. Animal caps were treated with 0.1 or 1 M atRA for 4 h at room temperature. RA-responsive genes lhx1, gbx2, hoxd1, and cdx4 were
induced by atRA treatment in a dose-dependent manner. Depletion of Hspa5 leads to decreased induction of these genes. C, real time PCR shows that HSPA5
shRNA effectively reduces HSPA5mRNAlevels in human HEK293T cells. Expression of HSPA5 in cells transfected with either control shRNA or HSPA5 shRNA was
normalized to the control cells. D–G, endogenous HSPA5 expression is reduced in HSPA5 shRNA-infected (D and E) or HSPA5 siRNA-infected (F and G) HEK293T
cells. The expression of HSPA5 was detected by Western blotting. -Tubulin was used as a loading control (D and F). The quantified HSPA5 signal in either
HSPA5 shRNA-infected cells or control shRNA-infected cells was normalized to the -tubulin signal. The relative expression was normalized to that of control
cells (E and G). The intensity of Western blot signals was quantified by a GS-800 calibrated imaging densitometer. H and I, depletion of HSPA5 by either Hspa5
shRNA (H) or Hspa5 siRNA (I) reduces the luciferase activity of RA luciferase reporter gene in HEK293T cells. atRA treatment enhances the luciferase activity,
whereas HSPA5 shRNA reduces the luciferase activity in a dose-dependent manner. BMS453, an RA signaling inhibitor, also inhibits the luciferase activity of RA
luciferase reporter gene.DMSOtreatment was used as a control. The asterisks indicate the statistically significant difference. con, control; RLU, relative luciferase
units. Error bars represent S.D. Image published in: Shi W et al. (2015) Copyright © 2015. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |