Skip to main content
. 2022 Jun 6;11:e66005. doi: 10.7554/eLife.66005

Figure 3. Sensitivity of chick and duck cells to rTGFβ1 and effects of TGFβR1 and SMAD3 inhibition on RUNX2 and MMP13.

(A) pSMAD3 protein levels in cells treated for 2 hr with 5 ng/ml rTGFβ1 (dark gray) in chick (blue) and duck (yellow) cells show a significant induction in chick (n=8). (B) Chick and duck cells treated with rTGFβ1 for 1–24 hr. Chick Runx2 mRNA increases with rTGFβ1 treatment at 3 and 6 hr and at 24 hr, while duck Runx2 does not increase until 24 hr. In rTGFβ1 treated cells, duck have significantly lower Runx2 at every time point compared to chick (n=8). (C) Chick Mmp13 mRNA increases with rTGFβ1 treatment at 6 hr and at 24 hr, while duck Runx2 does not increase until 24 hr. In rTGFβ1 treated cells, duck have lower Mmp13 at every time point compared to chick (n=8). (D) MMP13 protein in cells treated for 24 hr with rTGFβ1 shows an induction in chick but no response in duck (n=8). (E) RUNX2 protein levels in chick cells treated for 24 hr with rTGFβ1 (dark gray), TGFβR1 inhibitor (medium gray), a combination of both rTGFβ1 and TGFβR1 inhibitor (black), SMAD3 inhibitor (white), and a combination of both rTGFβ1 and SMAD3 inhibitor (light gray). RUNX2 protein increases with rTGFβ1, but when rTGFβ1 is combined with either a TGFβr1 or a SMAD3 inhibitor, there is a significant decrease compared to rTGFβ1 alone (n=12). (F) MMP13 protein increases with rTGFβ1 treatment, but when rTGFβ1 is combined with either a TGFβR1 or a SMAD3 inhibitor, there is a significant decrease compared to rTGFβ1 alone (n=12). * denotes significance from control p≤0.05, ** denotes significance from control p≤0.01, *** denotes significance from control p≤0.001, # denotes significance from rTGFβ1, † denotes significance between chick and quail, and ‡ denotes significance between chick and duck.

Figure 3.

Figure 3—figure supplement 1. Representative western blot images.

Figure 3—figure supplement 1.

Protein levels for (A) phosphorylated (p) SMAD3 (50 kDa) and (B) MMP13 (54 kDa) in chick (DF-1) and duck (CCL-141) cells treated with 5 ng/ml rTGFβ1 for 2 hr. β-Actin (42 kDa) is used as a loading control (n=8).
Figure 3—figure supplement 1—source data 1. Western blot images for pSMAD and β-Actin.
Figure 3—figure supplement 1—source data 2. Western blot images for MMP13 and β-Actin.
Figure 3—figure supplement 2. Sensitivity of chick and duck cells to rTGFβ1 and effects on TGFβ target genes.

Figure 3—figure supplement 2.

A) Pai1 in chick (blue) and duck (yellow) cells treated for 1–24 hr with rTGFβ1 (dark gray). rTGFβ1 induces chick Pai1 mRNA at 24 hr, while duck Pai1 increases at 1 hr and 24 hr (n=8). (B) Mmp2 increases in chick cells following rTGFβ1 treatment at 6 and 24 hr, while in duck cells Mmp2 is induced at 1 and 24 hr (n=8). * denotes significance from HH31 within each group p≤0.05, ** denotes significance from HH31 within each group p≤0.01, # denotes significance between quail and duck at same stage, † denotes significance between chick and quail, and ‡ denotes significance between chick and duck.
Figure 3—figure supplement 3. Representative western blot images.

Figure 3—figure supplement 3.

Protein levels for (A) RUNX2 (55 kDa) and (B) MMP13 (54 kDa) in chick cells treated for 24 hr with rTGFβ1, TGFβR1 inhibitor, a combination of both rTGFβ1 and TGFβR1 inhibitor, SMAD3 inhibitor, and a combination of both rTGFβ1 and SMAD3 inhibitor (n=8). β-Actin (42 kDa) is used as a loading control.
Figure 3—figure supplement 3—source data 1. Western blot images for RUNX2 and β-Actin.
Figure 3—figure supplement 3—source data 2. Western blot images for MMP13 and β-Actin.