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2NCOMMS-18-06274-A Revise Supplementary Figure Supplementary Information FoxK1 and FoxK2 are Components in Insulin Regulation of Cellular and Mitochondrial Metabolism Sakaguchi et al a ProteomicsProteomics screening screening of IR/IGF1Rof IR/IGF1R binding binding proteins proteinsin the in insulinthe insulin dependent DepenDent manner manner Total: 1469proteins 5 quantified peptide: 707 proteins (Including IRS1 and IRS2) (IR+/IR-1.5, IGF1R+/IGF1R-1.5, IR/IGF1R+/ IR/IGF1R-1.5 ) IGF1R/IR+/ IGf1R/IR-1.5 ) Total 11 proteins IR +/- 2 1.5 3 IGF1R +/-1.5 11 Common proteins →protein 5 FoxK1 (Forkhead box protein K1) 3 IR/IGF1R +/- 1.5 IGF1R/IR +/-1.5 b 5000 4000 3000 2000 1000 FoxK1 Expression FoxK1 … 0 … TA SCF EDL PGF liver Skin Lung Heart RETF BATF Testis Uterus Spleen Soleus Kidney Bladder Stomach Pancreas Cerebellum Quadriceps Epidydimus Gall bladder Hippocampus Adrenal gland Hypothalamus Small intestine Raphe nucleus Gastrocnemius Nucleus tractus Substantia nigra Prefrontal cortex Caudate putamen Ventral tegmental c Nucleus accumbens 3000 2000 Expression 1000 FoxK2 FoxK2 … … 0 … TA SCF EDL PGF liver Skin Lung Heart RETF BATF Testis Uterus Spleen Soleus Kidney Bladder Nucleus Stomach Pancreas Cerebellum Quadriceps Epidydimus Gall bladder Hippocampus Adrenal gland Hypothalamus Small intestine Raphe nucleus Gastrocnemius Nucleus tractus Substantia nigra Prefrontal cortex Caudate putamen Ventral tegmental Sakaguchi et al., Supplementary Figure 1 d DifferentiateD brown adipocytes e Astrocytes Cytoplasm Nucleus Cytoplasm Nucleus Insulin 0 5 10 30 0 5 10 30 min Insulin 0 5 10 30 0 5 10 30 min FoxK1 FoxK1 75 75 75 FoxO1 75 FoxO1 75 75 LaminA/C LaminA/C 37 37 GAPDH GAPDH Supplementary Figure 1. FoxK1 is a novel component of IR- and IGFR-mediated signaling complex. (a) FoxK1 is iDentifieD by the proteomics screening of IR/IGF1R binDing proteins. (b) FoxK1 and (c) FoxK2 expression (qPCR) from 34 tissues from 3-month-olD C57BL/6J mice (All Data are representeD as mean ± SEM, n=3). Data were normalizeD to Tbp. Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from DifferentiateD brown preaDipocytes (d) or astrocytes (e) before and after 100 nM insulin stimulation for inDicateD times. Sakaguchi et al., Supplementary Figure 1 a b Insulin - - - - + + + + WT (Brown preaDipocyte) 75 Insulin 0 1 5 10 30 60 min FoxO1 Cytoplasm Cytoplasm FoxK1 FoxK1 75 75 75 37 FoxO1 GAPDH 37 GAPDH 75 FoxO1 FoxK1 Nucleus Nucleus 75 75 FoxK1 FoxO1 75 75 75 LaminA/C Lamin A/C 75 FoxO1 Chromatin FoxK1 75 75 LaminA/C exp 1 2 3 4 1 2 3 4 Supplementary Figure 2. Insulin-induced FoxK1 translocation is reciprocal to FoxO1. (a) Subcellular fractions of cytoplasm, nucleus anD chromatin were prepareD from DKO brown preaDipocytes reexpressing the IR without (0 min) anD with 100 nM insulin stimulation for 30 min. FoxO1 anD FoxK1 in each fraction were assesseD by western blotting, as were markers for Different fractions, cytosol (GAPDH), nuclear anD chromatin (Lamin A/C) (n = 4). (b) Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from wilD-type brown preaDipocytes before and after 100 nM insulin stimulation for 30 min. Sakaguchi et al., Supplementary Figure 2 a Vehicle MK2206 U0126 Insulin - - - + + + - - - + + + - - - + + + 75 FoxO1 Cytoplasm FoxK1 75 37 GAPDH 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 exp 75 FoxO1 Nucleus FoxK1 75 75 LaminA/C b vehicle LY294002 Insulin IGF1 PDGF EGF Insulin IGF1 PDGF EGF Insulin - + - + - + - + - + - + - + - + FoxK1 75 Nucleus 75 LaminA/C c ★★★ ★★★ ★★ 3.0 ★★★ 2.5 2.0 1.5 FoxK1 Intensity (AU) 1.0 0.5 0.0 Relative Nuclear - + + + + + + + + Insulin IGF1 Sakaguchi et al., Supplementary Figure 3 d e Insulin - + + + + + IGF1 - + + + + + FoxK1 FoxK1 75 75 lysate 37 GAPDH lysate 37 GAPDH Cytoplasm Cytoplasm Cytoplasm Cytoplasm 100 FoxK1 FoxK1 75 lysate lysate 75 75 Nucleus Nucleus Nucleus Nucleus LaminA/C LaminA/C p-Akt p-Akt (S473) 50 (S473) 50 p-ERK1/2 p-ERK1/2 (T202/Y204) (T202/Y204) lysate lysate 37 37 Whole cell cell Whole Whole cell cell Whole T-Akt T-Akt 50 50 f Supplementary Figure 3. Insulin-induced FoxK1 translocation is regulated by PI3K- Akt rather than ERK MAPK pathway. (a) Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from IR-expressing brown preaDipocytes before and after 10 nM insulin stimulation for 30 min in the presence or absence of the Akt inhibitor MK2206 (5 μM) or the MEK1/2 inhibitor U0126 (20 μM) (n = 4). (b) Immunoblotting of FoxK1 in nuclear fraction extracteD from WT-brown preaDipocytes before anD after stimulation with insulin, IGF1, PDGF anD EGF at 15 min in the presence or absence of the PI3K inhibitor (LY-294002). (c) Relative FoxK1 fluorescence intensity in nuclear (DAPI+) area quantifieD by Image J in Figure 2D. All Data are representeD as mean ± SEM. (P < 0.05; **, P < 0.01; ***, P < 0.001) (n = 4). (d,e) Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from AML12 cells before or after stimulation with 100 nM insulin anD IGF1 for 30 min in the presence or absence of the Akt inhibitor MK2206 (5 μM) or the MEK1/2 inhibitor U0126 (20 μM). (e) Comparison of the phosphorylation clusters between FoxK1 anD FoxK2 after Insulin/IGF1 stimulation. The significant increase after Insulin/IGF1 stimulation shown in reD anD the Decrease shown in blue. Sakaguchi et al., Supplementary Figure 3 a Vehicle Rapamycin CHIR99021 Insulin - - - + + + - - - + + + - - - + + + FoxK1 80 Cytoplasm FoxK2 65 GAPDH 35 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 exp FoxK1 80 Nucleus FoxK2 80 LaminA/C 50 b Control GSK3α KD c Control GSK3β KD exp 1 2 3 4 1 2 3 4 exp 1 2 3 4 1 2 3 4 55 GSK3α 50 GSK3β 37 GAPDH GAPDH 34 Cytoplasm Nucleus d Control GSK3α/β DKD e Insulin - - - - + + + + - - - - + + + + Cytoplasm GSK3β GSK3β - - FoxK1 Control DKD GSK3α/β +HA Control DKD GSK3α/β +HA 75 DKD GSK3α/β DKD GSK3α/β 37 Insulin - + - + - + - + - + - + GAPDH FoxK1 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 exp 75 Nucleus FoxK1 37 75 GAPDH LaminA/C 75 LaminA/C 75 f Insulin, 15 min g Insulin, 15 min IRb IRb 95 95 FoxK1 95 95 FoxK1 p-IRb 95 p-IRb 95 IRb 95 95 FoxK1 FoxK1 43 95 b-Actin 43 b-Actin Sakaguchi et al., Supplementary Figure 4 h Cytoplasm Nucleus Flag-FoxK1 WT Flag-FoxK1 Flag-FoxK1 WT Flag-FoxK1 S402A/S406A/S454A/S458A S402A/S406A/S454A/S458A Insulin - - - + + + - - - + + + - - - + + + - - - + + + 95 Flag 72 Lamin A/C GAPDH 34 Supplementary Figure 4. Insulin-induced FoxK1 translocation is regulated by not only mTOR but also GSK signaling. (a) Nuclear cytoplasmic fractionation anD immunoblotting for FoxK1 anD FoxK2 extracteD from the AML12 cells before anD after 100 nM insulin at 30 min the presence or absence of 100 nM rapamycin (mTOR inhibitor) or/and 10 μM CHIR99201 (GSK3 inhibitor). GAPDH is cytosolic marker anD Lamin A/C is nuclear marker (n = 3). Immunoblotting Control (NS siRNA), GSK3α-knockdown (GSK3α KD) (b) and GSK3β-knockdown (GSK3β KD) (c) cells by siRNAs with anti-GSK3α anD GSK3β antiboDies (n = 4). (d) Nuclear anD cytoplasmic fractionation anD immunoblotting of FoxK1 extracteD from the Control (NS siRNA) or GSK3α/β Double knockdown cells before (0 min) and after 100 nM insulin stimulation for 30 min. (n = 4). (e) Nuclear anD cytoplasmic fractionation anD immunoblotting of FoxK1 extracteD from the Control (NS siRNA) , GSK3α/β DKD cells anD re-expresseD with HA-GSK3β in GSK3α/β DKD cells. before (0 min) and after 100 nM insulin stimulation for 30 min.(f) Immunoblotting with anti- IRβ, p-IRβ anD FoxK1 antiboDy in lysates extracteD from AML12 cells which were immunoprecipitateD with p-Tyr antiboDy following insulin stimulation. (g) Immunoblotting with anti-IRβ, p-IRβ anD FoxK1 antiboDy in lysates extracteD from liver samples which were immunoprecipitateD with p-Tyr antiboDy following insulin stimulation via vena cava injections. (h) Nuclear anD cytoplasmic fractionation anD immunoblotting of Flag extracteD from the overexpresseD 3XFlag-FoxK1 wilD type (WT) cells or overexpresseD 3XFlag- FoxK1 S402A/S406A/S454A/S458A mutant cells before (0 min) anD after 100 nM insulin stimulation for 30 min. Sakaguchi et al., Supplementary Figure 4 a Control FoxK1 OE FoxK2 OE b FoxK1 3.0 1 2 3 1 2 3 1 2 3 exp ★ ★ - + - + - + - + - + - + - + - + - + Insulin 2.5 Flag 2.0 75 1.5 FoxK1 95 1.0 FoxK2 72 0.5 37 GAPDH Control - Control + FoxK1 OE- FoxK1 OE+ FoxK2 OE- FoxK2 OE+ 0.0 - + - + - + d Control FoxK1 OE FoxK2 OE 1 2 3 1 2 3 1 2 3 exp - + - + - + - + - + - + - + - + - + Insulin p-IR/IGF1R c FoxK2 75 10.0 ★★★ IRb 75 8.0 ★ p-IRS-1 (Y612) 150 6.0 IRS-1 150 4.0 p-Akt (S473) 50 2.0 50 Akt p-ERK1/2 0.0 Control - Control + FoxK1 OE- FoxK1 OE+ FoxK2 OE- FoxK2 OE+ 37 (T202/Y204) - + - + - + 37 ERK1/2 37 p-S6 (S235/236) 37 S6 p-IR/ /IRb p-IRS-1 (Y612) p-Akt (S473) p-ERK1/2 (T202/Y204) p-S6 (S235/236) e f g h 1.5 ★ i 1.5 ★★★ 1.5 1.5 1.5 expression Relative Relative expression Relative expression Relative expression Relative Relative expression Relative NS NS 0.05 1.0 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 0.5 Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + 0.0 Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + 0.0 0.0 0.0 0.0 - + - + - + Insulin - + - + - + - + - + - + - + - + - + - + - + - + Supplementary Figure 5.
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