Fig 2: USP4 deubiquitinates PDK1 in vivo and in vitro. A: HEK293T cells were co-transfected with V5-tagged PDK1 and a DUB cDNA library. PDK1 was immunoprecipitated with anti-V5 beads and the Ub-PDK1 was detected using a V5 antibody. B: HEK293T cells were co-transfected as indicated. Proteins were immunoprecipitated with anti-V5 beads and immunoblotted with anti-V5 antibody. Whole cell lysates were probed with a MYC antibody to verify the expression of USP4 constructs. C: HEK293T cells were transfected with indicated DUBs. Endogenous PDK1 was immunoprecipitated and immunoblotted with a PDK1 antibody. Whole cell lysates were probed with V5 and FLAG antibodies. D: Quantification of four independent experiments as in C. Indicated are the mean and standard error of the mean and three asterisks indicate T-test p value<0.001. E: HEK293T cells were transfected as indicated and USP4 was immunoprecipitated using a MYC antibody. Immunoprecipitations were incubated in vitro with purified HA-tagged PDK1/Ub-PDK1 as a substrate and immunoblotted with anti-HA antibody. The expression of the USP4 constructs was verified with anti-MYC antibody.

Fig 3: USP4 and PDK1 interact and co-localize at the plasma membrane. A: HEK293T cells were transfected as indicated and PDK1 was immunoprecipitated with anti-V5 beads. Immunoprecipitations (IP) and input were probed with anti-V5 and MYC antibodies. B: Confocal images from transfected U2OS cells stained with V5-PDK1 (green) and MYC-USP4 (red) antibodies. DNA was visualized by DAPI.

Fig 4: CAPE and CAPPE inhibited the proliferation of human CRC HCT-116 cells through the modulation of the PI3K/Akt, AMPK and mTOR signaling pathways.Human CRC HCT-116 cells were treated with either CAPE or CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in 10% FBS RPMI-1640 for 24 h. (A) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against N-cadherin, PTEN, anti-phosphorylation PDK1 (S241), total-PDK1, anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation GSK3a (S21), total- GSK3a, anti-phosphorylation GSK3ß (S9), total- GSK3ß, anti-phosphorylation FOXO3 (T32), total- FOXO3, total- TSC1, total- TSC2, total- LKB1, total- 14-3-3, anti-phosphorylation AMPKa (T172), total-AMPKa, anti-phosphorylation m-TOR (S2448), total-m-TOR, anti-FASN and ß-actin as described under Materials and Methods. The levels of detection represent the amounts of each protein in the cytoplasm of HCT-116 cells. The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The immunoreactive bands are noted with an arrow. The mean integrated densities of these proteins adjusted with the control protein are shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk indicates a significant difference compared to the CAPE- or CAPPE-untreated control group, respectively (P<0.05). (B) The measurement of cellular ATP was performed as described under Materials and Methods. Data represent the percentage of cellular ATP levels in the CAPE- or CAPPE-treated human CRC HCT-116 cells. A single or double asterisk indicates a significant difference compared to the CAPE- or CAPPE-untreated control group, respectively (P<0.05).

Fig 5: Involvement of PDK1 in the induction by TSH of IL-6.(A) Cultures were untreated (control) or bTSH (5 mIU/ml) was added to medium for 30 min. Cellular protein was subjected to Western blot analysis for PDK1 and pPDK1 and re-probed for ß-actin. Results are representative of three separate experiments performed. Densitometric analysis; pPDK1, control fibroblasts, 16.25±2.85 AU; plus bTSH, 42.7±6.54 AU; control fibrocytes, 64.5±2.67 AU; plus TSH, 78.5±3.8 AU. In 3 separate experiments, TSH increased pPDK1 levels by 2.6±0.3-fold and 1.2±0.3-fold in fibroblasts and fibrocytes, respectively. (B) Sub- confluent cultures were transfected with either control siRNA or one targeting PDK1, followed by 48 h incubation. Cultures were treated with nothing or bTSH (5 mIU/mL) for 16 h, media collected and subjected to IL-6-specific ELISA and cell layers analyzed for protein content. Data are expressed as the mean ± SD of three independent determinations. Inset: Cell layers were subjected to Western blot analysis for PDK1 after transfection with control siRNA or that targeting PDK1. In 3 separate experiments, PDK1 siRNA reduced TSH-induced IL-6 levels by 73±4% in fibroblasts and 73±5% in fibrocytes. (C) Orbital fibroblasts, in this case from a patient with TAO, were transfected with PDK1siRNA while fibrocytes were treated with OSU-03012 (5 µM) for 6 h. Cultures were treated as indicated (bTSH, 5 mIU/mL) for 30 min. Cellular protein was subjected to Western blot analysis of PKCµ and pPKCµ in fibroblasts (left panel) and PKCßII and pPKCßII in fibrocytes (right panel). (D) Confluent cultures were pre-treated without or with OSU-03012 (5 µM) for 6 h, then treated with nothing (control) or bTSH (5 mIU/ml) for 30 min. Cellular proteins were subjected to Western blot analysis probing with AKT and pAKT antibodies. Inhibition of TSH-dependent pAKT by OSU-03012 in 3 separate experiments was 14.4±1.2% and 2.5±0.6% in fibroblasts and fibrocytes, respectively.