Fig 2: Dysregulation of IGF-1R/ERK signaling in Gigyf1 deficiency mice.(A) Immunoblots of pIgf-1r, Igf-1r, pErk1/2, Erk1/2, p27, cyclin D1, and Gigyf1 in lysates from brain cortical tissue of Gigyf1fl/fl, cHET and cKO mice at E14.5. The relative levels of pIgf-1r/total Igf-1r, pErk-1r/total Erk, p27, and cyclin D1 were quantified by densitometry and compared using 1-way ANOVA. (B) Neurosphere formation assay. Neural progenitor cells are derived from Gigyf1fl/fl, cHET and cKO embryos. Representative images of Gigyf1fl/fl, cHET and cKO neurosphere are shown. Scale bar: 10 μm. (C) The diameters of Gigyf1fl/fl, cHET, and cKO neurospheres were calculated and compared. Experiments were performed for 3 trials and the statistics are based on the average of each condition from different trials. Statistical data are analyzed using 1-way ANOVA and 2-tailed t test. All data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: GIGYF1 KO disrupts IGF-1R/ERK pathway by regulation of IGF-1R recycling.(A) Coimmunoprecipitation assay for GIGYF1 and IGF-1R in HEK293T cells. (B) Double immunofluorescence of GIGYF1 and IGF-1R in HeLa cells. Scale bars: 10 μm. Inset scale bars: 5 μm. (C) Immunoblots of the whole cell lysate showing levels of pIGF-1R, IGF-1R, pERK1/2, ERK1/2, pAkt, and Akt at different duration of IGF-1 stimulation. Statistical data were analyzed using 2-way ANOVA. (D) Immunoblots of the whole-cell lysates showing levels of pERK1/2, ERK1/2 in HEK293T GIGYF1 KO cells expressing mock empty vector (pCAGGS-IRES-GFP) or HA-GIGYF1. The relative levels of pERK1/2 to ERK1/2 were quantified by densitometry and analyzed using 2-way ANOVA. (E) Immunoblot of pERK1/2 and ERK1/2 in the whole-cell lysates at 7 minutes of IGF-1 stimulation. Statistic data were analyzed using 1-way ANOVA. (F) Immunoblots of biotin-labelled IGF-1R, total IGF-1R and TMEM98. Total IGF-1R-α levels of unbiotinylated cells were determined. The protein levels of surface IGF-1R, surface IGF-1R/total IGF-1R, and total IGF-1R were quantified by densitometry from 3 biological replicates. Statistical data were analyzed using 2-tailed Student’s t tests. (G) Immunoblots of biotin-labeled IGF-1R and total IGF-1R at different conditions in a surface biotinylation recycling assay. GIGYF1 KO HEK293T cells and control HEK293T cells (lanes 2–8) were surface labeled with sulfo-NHS-S-S-biotin. Cells (lanes 3–8) were incubated to endocytosis. The remaining surface biotin was cleaved with glutathione cleavage buffer (lanes 2–8). Cells were incubated for a second time to recycle (lanes 4–5 and 7–8), then the surface biotin was stripped for a second time (lanes 3–4 and 6–7). Lanes 5 and 8 were incubated to recycle without a second cleavage. The stage of biotinylation recycling assay for each lysate is indicated with + and –. (H) Working model of GIGYF1 regulation of IGF-1R/ERK pathway. All data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See complete unedited blots in the supplemental material.

Fig 4: Female mice with the IGF1RhR407H genotype showed alterations in IGF1R expression levels. A. Serum IGF1 levels in female WT and IGF1RhR407H (mutant) mice at 6 months of age (n=4–5). B-E. Western blotting was performed to measure the phosphorylation levels of IGF1R, AKT and the total IGF1R expression in heart (B), kidney (C), pancreas (D), and skeletal muscle (E) tissues (n=4 mice per genotype). The phosphorylated to total protein ratios and IGF1R total protein expression, as determined by densitometry, are shown on the right (B-E). IGF1R expression levels were normalized to each individual actin. All data represent the mean ± SEM. *p < 0.05, **p < 0.01, statistically significant difference between WT and IGF1RhR407H mice

Fig 5: PINCH-1 regulates IGF-1R expression and cell proliferation in the epidermis in vivo. (A) Mating strategy for generating the P1-K5 and control mice. (B) Photographs of the P1-K5 and control mice at 1 and 5 weeks of age. (C) The genotypes of the P1-K5 (left) and PINCH-1 flox/+ mice (right) were analyzed by PCR as described in the Methods. (D) Epidermal lysates (Epi) from the control and P1-K5 mice at 65 days of age were analyzed by WB with Abs for PINCH-1 and GAPDH (as a control). (E) The lysates of back skin tissues from the control and P1-K5 mice at 5 weeks of age were analyzed by WB with Abs for IGF-1R, PINCH-1 or tubulin (as a control). (F) The IGF-1R levels in the skin tissues of P1-K5 mice were quantified and compared to that of the control mice (normalized to 1, n = 3). (G-J) The back skin tissues from the control and P1-K5 mice treated with DMBA/TPA for 20 weeks were immunofluorescently stained with DAPI (blue) and Abs for IGF-1R (red) and K5 (green) (G) or Abs for Ki67 (red) and K5 (green) (I). Scale bars = 100 μm. (H) The immunofluorescence intensities of IGF-1R in the skin tissue sections of the P1-K5 mice were quantified and compared to those in the control mice (normalized to 1, n = 3). (J) The number of Ki67 positive cells in the skin tissue sections of P1-K5 mice were quantified and compared to those in the control mice (n = 3). Data are presented as mean ± SEM using t-test analysis, **P < 0.01.