Tgif1-deficiency impairs cytoskeletal architecture in osteoblasts by activating PAK3 signaling
- Molecular Skeletal Biology Laboratory, Department of Trauma Surgery and Orthopedics, University Medical Center Hamburg-Eppendorf, Germany
- Institute of Musculoskeletal Medicine, LMU University Hospital, LMU Munich, Germany
- Musculoskeletal University Center Munich, LMU University Hospital, LMU Munich, Germany
Figures
Figure 1 with 1 supplement
Loss of TG-interacting factor 1 (Tgif1) reduces osteoblast size in vivo and impairs osteoblast adhesion and migration in vitro.
(A) Representative images of the femora from 12-week-old Tgif1+/+ (n=4) and Tgif1-/- (n=4) mice stained with Toluidine blue. (B) Femora of 12-week-old Dmp1-Cre-;Tgif1fl/fl (n=4) and Dmp1-Cre+;Tgif1fl…
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Figure 1—source data 1
Numerical data related to Figure 1D, E, F, G and H.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Migration of calvarial osteoblasts obtained from neonatal Tgif1+/+ (n=20) and Tgif1-/- (n=20) mice was analyzed using live cell imaging.
The direction and distance of individual cell migration are visualized.
Figure 2 with 1 supplement
TG-interacting factor 1 (Tgif1)-deficient osteoblasts are impaired to spread and form focal adhesions.
(A, B) Calvarial osteoblast obtained from Tgif1+/+ and Tgif1-/- mice adhered on Col-I coated slides for 20, 40, and 60 min. (A) Cell spreading was visualized by Calcein-AM. (B) The number of round …
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Figure 2—source data 1
Numerical data related to Figure 2D, E, G and H.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Representative image of an immunoblot demonstrating the expression of TG-interacting factor 1 (Tgif1) in OCY454 cells transfected with scrambled control siRNA (Scr) or siRNA against Tgif1 (siTgif1).
Actin was used as a loading control.
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Figure 2—figure supplement 1—source data 1
Original files for the western blot analysis in Figure 2—figure supplement 1 (anti-Tgif1 and anti-actin).
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF containing Figure 2—figure supplement 1A and original scans of the relevant western blot analysis (anti-Tgif1 and anti-actin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig2-figsupp1-data2-v1.zip
Figure 3 with 3 supplements
Silencing of p21-activated kinase 3 (PAK3) restores cell spreading and focal adhesion formation in TG-interacting factor 1 (Tgif1)-deficient cells.
(A) Pak3 mRNA expression in OCY454 cells transfected with siRNA targeting Tgif1 (siTgif1) or scrambled control (scr) siRNA before (0) and after 20 and 60 min of spreading on Col-I coated slides. …
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Figure 3—source data 1
Numerical data related to Figure 3A, C, D, E, G and H.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-data1-v1.xlsx
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Figure 3—source data 2
Original files for the western blot analysis in Figure 3B (anti-PAK3 and anti-actin).
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-data2-v1.zip
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Figure 3—source data 3
PDF containing Figure 3B and original scans of the relevant western blot analysis (anti-PAK3 and anti-actin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-data3-v1.zip
Figure 3—figure supplement 1
TG-interacting factor 1 (Tgif1)-deficiency does not alter the abundance or activation of the FA components and Tgif1 does not co-localize with FA complexes.
(A) Osteoblasts adhered on Col-I coated slides for 0, 20, or 60 min. Representative images of immunoblots demonstrating the abundance of p-FAK, FAK, p-p38, p38, p-Paxillin, Paxillin, p-src, src, …
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Figure 3—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 3—figure supplement 1A (anti-pFAK, anti-FAK, anti-pp38, anti-p38, anti-ppaxillin, anti-paxillin, anti-psrc, anti-src, anti-integrin β1, anti LRG5, anti-talin, anti-actin).
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF containing Figure 3—figure supplement 1A and original scans of the relevant western blot analysis (anti-pFAK, anti-FAK, anti-pp38, anti-p38, anti-ppaxillin, anti-paxillin, anti-psrc, anti-src, anti-integrin β1, anti LRG5, anti-talin, anti-actin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Lack of TG-interacting factor 1 (Tgif1) does not alter the expression of Cdcs, Pak11, Pak2, or Pak4.
OCY454 cells were transfected with a siRNA targeting Tgif1 (siTgif1) or scramble control (scr) prior to adherence on Col-I coated slides for 20 and 60 min. Gene expression was quantified by qPCR for …
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Figure 3—figure supplement 2—source data 1
Numerical data related to Figure 3—figure supplement 2A-G.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-figsupp2-data1-v1.xlsx
Figure 3—figure supplement 3
Tgif1 binding site on the PAK3 promoter and expression of PAK3 and Tgif1.
(A) Schematic of the conserved predicted TG-interacting factor 1 (Tgif) binding site on the rat and mouse p21-activated kinase 3 (Pak3) promoters. (B) OCY454 cells were transfected with siRNA …
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Figure 3—figure supplement 3—source data 1
Original file for the western blot analysis in Figure 3—figure supplement 3 (anti-PAK3, anti-Tgif1, and anti-actin).
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-figsupp3-data1-v1.zip
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Figure 3—figure supplement 3—source data 2
PDF containing Figure 3—figure supplement 3 and original scans of the relevant western blot analysis (anti-PAK3, anti-Tgif1, and anti-actin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig3-figsupp3-data2-v1.zip
Figure 4
TG-interacting factor 1 (Tgif1) expression is increased during cell spreading via ERK and AP1 signaling pathways.
(A) Quantification of Tgif1 mRNA expression and (B) representative images of immunoblots of Tgif1 expression in calvarial osteoblasts before (0 min) and after 20 and 60 min of spreading. Actin was …
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Figure 4—source data 1
Numerical data related to Figure 4A, C, E, G, H, I and J.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig4-data1-v1.xlsx
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Figure 4—source data 2
Original files for the western blot analysis in Figure 4B (anti-Tgif1 and anti-actin).
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig4-data2-v1.zip
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Figure 4—source data 3
PDF containing Figure 4B and original scans of the relevant western blot analysis (anti-Tgif1 and anti-actin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig4-data3-v1.zip
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Figure 4—source data 4
Original files for the western blot analysis in Figure 4D (anti-Tgif1 and anti-actin), anti-pERK1/2, anti-ERK1/2, and anti-tubulin.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig4-data4-v1.zip
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Figure 4—source data 5
PDF containing Figure 4D and F and original scans of the relevant western blot analysis (anti-Tgif1 and anti-actin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig4-data5-v1.zip
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Figure 4—source data 6
Original files for the western blot analysis in Figure 4F (anti-pERK1/2, anti-ERK1/2, and anti-tubulin).
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig4-data6-v1.zip
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Figure 4—source data 7
PDF containing Figure 4F and original scans of the relevant western blot analysis (anti-pERK1/2, anti-ERK1/2, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig4-data7-v1.zip
Figure 5
TG-interacting factor 1 (Tgif1) deficiency causes a reduced number and activity of osteoblasts during bone repair.
(A) Representative images of Toluidine blue-stained sections of bones from male Tgif1+/+ (n=9) and Tgif1-/- mice (n=5) that received an open fracture of the midshaft tibia at 10 weeks of age …
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Figure 5—source data 1
Numerical data related to Figure 5B, C, D and E.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig5-data1-v1.xlsx
Figure 6
PTH is impaired in activating quiescent bone surfaces in TG-interacting factor 1 (Tgif1)-deficient mice.
(A) Toluidine blue staining of tibiae from 12-week-old Tgif1+/+ and Tgif1-/- mice treated with vehicle (veh) or PTH. Quiescent surfaces are indicated by red arrows. Representative images are shown. …
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Figure 6—source data 1
Numerical data related to Figure 6B and D.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig6-data1-v1.xlsx
Figure 7
PTH promotes cell spreading via TG-interacting factor 1 (Tgif1)-PAK3 signaling.
(A) OCY454 cells were transfected with siRNA against Tgif1 (siTgif1) or scramble control siRNA (scr) and treated with vehicle (veh) or PTH for 4 hr. Cells were allowed to adhere on Col-I coated …
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Figure 7—source data 1
Numerical data related to Figure 7B, C and F.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig7-data1-v1.xlsx
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Figure 7—source data 2
Original files for the western blot analysis in Figure 7D (anti-PAK3, anti-Tgif1, and anti-actin).
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig7-data2-v1.zip
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Figure 7—source data 3
PDF containing Figure 7D and original scans of the relevant western blot analysis (anti-Tgif1, anti-actin, anti-pERK1/2, anti-ERK1/2, and anti-tubulin) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/94265/elife-94265-fig7-data3-v1.zip
