1. Requirement of Gαi1/3–Gab1 Signaling Complex for Keratinocyte Growth Factor– Induced PI3K–AKT–mTORC1 Activation 
Yi-ming Zhang, Zhi-qing Zhang, Yuan-yuan Liu, Xin Zhou, Xiao-hua Shi, Qin Jiang, Dong-li Fan, Cong Cao 
Journal of Investigative Dermatology 
Volume 135, Issue 1, Pages (January 2015)
DOI: /jid
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2. Figure 1
Gαi1 and Gαi3 are required for the activation of AKT-mTORC1 by keratinocyte growth factor (KGF) in mouse embryonic fibroblast (MEFs). (a) Wild type (WT) MEFs were infected with virus particles containing keratinocyte growth factor receptor (KGFR)-pSUPER-puro-retro vector for 2 days, followed by 1.0 μg ml−1 of puromycin selection to generate KGFR-expressing MEFs (termed MEFs/KGFR), and KGFR and glyceraldehyde-3-phosphate dehydrogenase mRNA expression was tested by reverse transcriptase–PCR. (b) KGFR, β-actin, and EGFR expression in MEFs, MEFs/KGFR (Clone-1/-2), and primary keratinocytes were tested by western blotting. (c–h) WT MEFs/KGFR, Gαi1 or Gαi3 single-knockout (SKO) MEFs/KGFR, and Gαi1/3 double knockout (DKO)-MEFs/KGFR were treated with indicated KGF (50 ng ml−1), EGF (50 ng ml−1, 10 min), fibroblast growth factor (FGF)-10 (25 ng ml−1, 10 min), basic fibroblast growth factor (bFGF) (25 ng ml−1, 10 min), or platelet-derived growth factor (PDGF)-BB (25 ng ml−1, 10 min), and listed proteins were analyzed by western blotting. Indicated proteins or protein phosphorylation was quantified (for e–h; n=3, same for all blot quantifications). *P<0.05. #P<0.05 versus Gαi1/3 DKO MEFs/KGFR group (e).
Journal of Investigative Dermatology  , DOI: ( /jid )
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3. Figure 2
Gαi1 and Gαi3 proteins are vital for the phosphorylation of AKT (473 S) and S6 (235/236 S) in response to keratinocyte growth factor (KGF) in keratinocyte growth factor receptor (KGFR)-expressing mouse embryonic fibroblast (MEFs). (a, b, f) Wild type (WT) MEFs/KGFR transfected with scramble small interfering RNA or Gαi1/3 small interfering RNAs were treated with KGF (50 ng ml−1), basic fibroblast growth factor (bFGF) (25 ng ml−1), fibroblast growth factor (FGF)-10 (25 ng ml−1), or platelet-derived growth factor (PDGF)-BB (25 ng ml−1) for 10 min, and western blotting was utilized to test indicated proteins. (c) Double knockout (DKO) MEFs/KGFR were transfected with the vector encoding WT or dominant-negative (DN) Gαi3 (G202T; 2 μg each), and KGF (50 ng ml−1)-induced signaling in DKO MEFs (no KGFR, first two lanes) and above MEFs/KGFR (last four lanes) was tested. (d) KGF (50 ng ml−1)-induced AKT activation in WT and Gαi2 KO MEFs/KGFR was treated. (e) WT MEFs were pretreated overnight with pertussis toxin (PTX, 100 ng ml−1) followed by indicated KGF treatment, and pAKT (473S) and AKT were detected by western blotting. Indicated proteins or protein phosphorylation was quantified. *P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
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4. Figure 3
Gαi1 and Gαi3 proteins are required for keratinocyte growth factor (KGF)-induced AKT-mTOR complex 1 (mTORC1) activation in primary keratinocytes. (a, e) KGF (50 ng ml−1)-induced signaling in stable primary keratinocytes expressing scramble or Gαi1/3 short hairpin RNAs or Gαi2 short hairpin RNA was tested. (b) Primary keratinocytes were introduced with anti-sense RNAs against Gαi1/3 or scramble RNA sequence, and KGF (50 ng ml−1)-induced signaling was tested in the above cells. (c) KGF (50 ng ml−1)-induced signaling in stable primary keratinocytes expressing empty vector or GFP-Tagged Gαi1/3 was detected. (d) KGF (50 ng ml−1, 10 min) or platelet-derived growth factor (PDGF)-BB (25 ng ml−1, 10 min)-induced AKT activation in stable primary keratinocytes expressing empty vector or GFP-dominant-negative (DN)-Gαi3 was shown. All protein phosphorylations in this figure were quantified. *P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Gab1, lying downstream of Gαi, is required for keratinocyte growth factor (KGF)-induced AKT activation. (a, b) KGF (50 ng ml−1)-induced signaling in keratinocyte growth factor receptor (KGFR)-expressing wild type (WT) or growth factor receptor binding 2-associated binding protein 1 (Gab1) KO mouse embryonic fibroblast (MEFs) was detected by western blotting. (c) KGFR-expressing WT and Gαi1/3 double knockout (DKO) MEFs were treated with KGF (50 ng ml−1), and pGab1 (627Y), Gab1, p-p85 (458Y), p85, and KGFR were tested. (d, e) KGF (50 ngml−1)-induced signaling in stable primary keratinocytes expressing scramble or Gab1 short hairpin RNA was tested. (f) Stable primary keratinocytes expressing scramble or Gαi1/3 short hairpin RNAs were treated with KGF (50 ng ml−1), and p-Gab1(627Y), Gab1, and Gαi1 were tested. (g) Primary keratinocytes were treated with KGF (50 ngml−1) for the indicated time, and the association between Gαi3, Gab1, and KGFR was tested by co-immunoprecipitation (Co-IP). (h) Stable primary keratinocytes expressing scramble or Gαi1/3 short hairpin RNAs were treated with KGF (50 ngml−1) for 2 minutes, and expression and association of KGFR-FGF receptor substrate 2 (FRS-2)-Gαi3-Gab1 were tested. All protein phosphorylations in this figure were quantified. *P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Increased expression of Gαi1/3 in human wounded skin and keloid skin tissues. (a, b) Gαi1/Gαi3 immunohistochemistry of the granulation tissue of healing skin or surrounding healthy skin from the same patient (male, 35 years old, multiple skin wounds developing for 1 month). (c, d) Gαi1/Gαi3 immunohistochemistry of the skin keloid tissue and surrounding healthy skin tissue from the same patient (male, 27 years old, keloids develop for 4 months). Similar results were seen in three independent patients. Bar=100 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
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7. Figure 6
Gαi proteins are important for keratinocyte growth factor (KGF)-dependent cell growth and migration in keratinocytes. Stable primary keratinocytes expressing scramble or Gαi1/3 short hairpin RNAs were treated with KGF (25/50 ngml−1) or platelet-derived growth factor (PDGF)-BB (25 ngml−1) for 24 hours, and cell proliferation and migration were detected by the BrdU incorporation assay (a), the /[H3] Thymidine DNA assay (b), and the “Transwell” assay (d), and expression of Gαi1, Gαi3, and β-actin (c and e) as well as of cyclin D1 and fibronectin (f) in the above cells was tested by western blotting. (g) The proposed signaling pathway of this study (see Discussion section). *P<0.05 (a, b, d).
Journal of Investigative Dermatology  , DOI: ( /jid )
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