Journal: Aging Cell

Article Title: Age‐associated cholesterol reduction triggers brain insulin resistance by facilitating ligand‐independent receptor activation and pathway desensitization

doi: 10.1111/acel.12932

Figure Lengend Snippet: Insulin signaling is impaired in old mice due to PI3K/Akt hyperactivation. (a) Insulin‐LTD is completely abolished in old mice (20–24 months old; n = 7) compared to adult mice (7–12 months old; n = 10). The graphic represents insulin‐LTD as fEPSP slope. Gray box indicates the time of insulin application. Representative analogue traces on the right were collected at the indicated time points. (b) Insulin receptor (IR) basal activity levels detected by Western blot using Phospho‐Tyrosine 1150/1151 antibody after total protein immunoprecipitation. (c) Western blots reflect IGF‐1R Phospho‐Tyrosine 1135/1136 basal levels after total protein immunoprecipitation. Old mice hippocampus shows higher basal activity levels of IR and IGF1‐R compared to adult mice. (d) Akt Phospho‐Serine 473 activating residue detected by Western blot in adult and old mice hippocampus at basal level. (e) Western blot analysis of GSK3β in hippocampal extracts from adult and old mice. Consistent with Akt hyperactivation, the old hippocampus presents high GSK3β inhibitory mark (Phospho‐Serine 9). (f, g) PI3K/Akt inhibition rescues insulin‐LTD in old mice. Graphics showing insulin‐LTD induction in hippocampal slices from old mice incubated with Wortmannin (f; 0.5 μM; n = 7) and Quercetin (g; 20 μM; n = 8). Black box indicates time of insulin application. Gray box indicates time of inhibitors application. Representative analogue traces below were collected at the indicated time points. Numbers in bars reflect number of independent experiments. Data are represented as mean ± SEM . t test for (b, c, d, e), one‐way ANOVA with post hoc Bonferroni's test for (a, f, g). The asterisks p values (* p < 0.05; ** p < 0.01; ns = not significant)

Article Snippet: The following antibodies were used for Western blot (WB) and immunoprecipitation (IP): anti‐α‐Tubulin (WB 1:10,000, Abcam, Cambridge, UK ref.: ab7291), anti‐β‐Actin (WB 1:20,000; Sigma‐Aldrich ref.: A5441), anti‐GAPDH (WB 1:20,000; Abcam, ref.: ab8245), anti‐Akt (WB 1:1,000; Cell Signaling, Danver, MA ref.: #9272), anti–Phospho‐Akt Serine 473 (WB 1:1,000, Cell Signaling ref.: #4060), anti‐GSK3 α/β (WB 1:1,000, Invitrogen, Carlsbad, CA ref.: 44–610), anti‐Phospho GSK3 α/β Serine 21/9 (WB 1:1,000; Cell Signaling ref.: #9331), anti‐IGF‐1 Receptor β (WB 1:1,000, IP 1:100; Cell Signaling ref.: #9750), anti‐Phospho IGF‐1 receptor β Tyr1135/116 (WB 1:1,000; Cell Signaling ref.: #3024), anti‐Insulin receptor β (IP 1:100, Santa Cruz, Dallas, TX ref.: sc‐57342), anti‐Insulin receptor β (WB 1:750, Santa Cruz, Dallas, TX ref.: sc‐711), anti‐Phospho insulin receptor β Tyr1150/1151 (WB 1:750; Millipore, Burlington, MA ref.: 04‐299), anti‐IRS‐1 (WB 1:1,000; BD Biosciences, Franklin Lakes, NJ ref.: 611394), anti‐Phospho IRS‐1 Serine 307 (WB 1:750; Abcam ref.: ab4865), anti‐Phospho IRS‐1 Serine 632 (WB 1:200; Cell Signaling ref.: #2388), anti‐Phospho IRS‐1 Serine 1101 (WB 1:1,000; Cell Signaling ref.: #2385), anti‐mTOR (WB 1:1,000; Cell Signaling ref.: #4517), anti‐Phospho mTOR Serine 2448 (WB 1:2,500; Cell Signaling ref.: #5536), anti‐p70S6K (WB 1:1,000; BD Biosciences ref.: 611260), anti‐Phospho p70S6K Threonine 389 (WB 1:750; Cell Signaling ref.: #9206), anti‐IRS‐2 (WB 1:500, Upstate 06‐506), anti‐Vinculin (WB 1:1,000; Millipore ref.: AB6039).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Residue, Inhibition, Incubation

Journal: Cerebellum (London, England)

Article Title: Development and Optimization of a Multilayer Rat Purkinje Neuron Culture

doi: 10.1007/s12311-022-01510-4

Figure Lengend Snippet: Optimal conditions for culture of rat Purkinje neurons. A Supplementation with IGF1 and progesterone induced a stable environment that resulted in high survival rates of Purkinje neurons, whereas PKC inhibition (with K252a) shaped dendritic tree development for E18 and P0 cultures or impacted survival for P10-derived culture. The K252a starting concentration depends on the tissue used to start the culture. During the washout phase ( DIV 22–28), no K252a is added to the media. At 28 DIV , concentrations of IGF1 and progesterone are reduced. The protocol allows growth of a stable Purkinje neuron culture for up to 6 months ( DIV 169) in 6- to 24-well formats. B The time point for placement of the Purkinje neuron layer depends on the age of tissue harvest. For E18, the highest survival was found when placed on a support layer of 14 DIV ; for P0, the support layer should be 21 DIV ; and for P10, the support layer should be 28 DIV

Article Snippet: The support cell layer growth media was supplemented with insulin (Invitrogen, #12,585,014; 1:250, stock 4 mg/mL), progesterone (Sigma, #P8783, 1:2000, stock 80 mM), insulin-like growth factor 1 (IGF1; Promokine, #E-60840, 1:40,000, stock 1 µg/µL), and protein kinase C inhibitor K252a (Alomone, # K-150; IC 50 25 nM).

Techniques: Inhibition, Derivative Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: IGF1R Is a Potential New Therapeutic Target for HGNET-BCOR Brain Tumor Patients

doi: 10.3390/ijms20123027

Figure Lengend Snippet: IGF2 and IGF1R are highly expressed in high-grade neuroepithelial tumor with BCOR alteration (HGNET-BCOR). ( A ) Protein sequence of the BCOR allele of patients P1, P2, and P3 carrying BCOR internal tandem duplications (ITD) compared to wild-type (wt) BCOR . ( B , C ) qRT-PCR analysis was performed using primers specific for IGF2 and IGF1R . Data are shown as the fold change of the expression in the tumor tissues of three HGNET-BCOR patients (P1, P2, and P3) and one hepatoblastoma patient (P4) with respect to the expression in normal liver (control). Expression analysis was done in triplicate on RNA extracted from formalin-fixed paraffin-embedded (FFPE) material. ( D ) qRT-PCR analysis was performed using primers specific for IGF2 and IGF1R , with RNA extracted from PhKh1 cells or frozen material from the metastasis of origin of PhKh1 cells. After normalization to the expression of the housekeeping gene HPRT1 , the relative quantification value was expressed as 2−ΔΔCt. The calibrator was calculated as the maximal number of cycles used in the PCR (40) minus the mean of the HPRT1 Ct values, resulting in a value of 19.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (MA, USA): GAPDH (14C10) (cat # 2118, 1:1000 dilution), Lamin B1 (D4Q4Z) (cat# 12586, 1:1000 dilution) (Cell Signaling Technology), IGF1 Receptor β (D23H3) XP® (cat # 9750, 1:1000 dilution), Phospho-IGF1 Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) (cat # 3021, 1:1000 dilution), AKT (cat # 9272, 1:1000 dilution), Phospho-AKT (Ser473) (cat # 9271, 1:1000 dilution).

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Formalin-fixed Paraffin-Embedded

Journal: International Journal of Molecular Sciences

Article Title: IGF1R Is a Potential New Therapeutic Target for HGNET-BCOR Brain Tumor Patients

doi: 10.3390/ijms20123027

Figure Lengend Snippet: Overview of chemotherapeutics and insulin-like growth factor receptor (IGF1R) inhibitors tested in vitro on PhKh1 cells. The IC50, class of the drug, its mechanism of action, and tumor entity generally treated with the drugs are indicated. FDA: Food and Drug Administration.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (MA, USA): GAPDH (14C10) (cat # 2118, 1:1000 dilution), Lamin B1 (D4Q4Z) (cat# 12586, 1:1000 dilution) (Cell Signaling Technology), IGF1 Receptor β (D23H3) XP® (cat # 9750, 1:1000 dilution), Phospho-IGF1 Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) (cat # 3021, 1:1000 dilution), AKT (cat # 9272, 1:1000 dilution), Phospho-AKT (Ser473) (cat # 9271, 1:1000 dilution).

Techniques: In Vitro

Journal: International Journal of Molecular Sciences

Article Title: IGF1R Is a Potential New Therapeutic Target for HGNET-BCOR Brain Tumor Patients

doi: 10.3390/ijms20123027

Figure Lengend Snippet: HGNET-BCOR is sensitive to IGF1R inhibition: ( A – C ) PhKh1 cells were treated with the indicated IGF1R inhibitors at concentrations from 1 nM to 100 μM. The logarithm of the molarity (M) is displayed on the X-axis. The percent of viable cells compared to the control treated with vehicle alone is shown on the Y-axis. The data were fitted to a sigmoidal dose–response curve using GraphPad software. A representative experiment of three independent experiments is shown. ( D ) PhKh1 and DAOY cells were incubated with a blocking anti-IGF1R antibody at the indicated concentration, and cell proliferation was measured. The percent of viable cells compared to the control treated with vehicle alone is shown on the Y-axis. Statistics were performed using Student’s t -test. A representative experiment of three independent experiments is shown.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (MA, USA): GAPDH (14C10) (cat # 2118, 1:1000 dilution), Lamin B1 (D4Q4Z) (cat# 12586, 1:1000 dilution) (Cell Signaling Technology), IGF1 Receptor β (D23H3) XP® (cat # 9750, 1:1000 dilution), Phospho-IGF1 Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) (cat # 3021, 1:1000 dilution), AKT (cat # 9272, 1:1000 dilution), Phospho-AKT (Ser473) (cat # 9271, 1:1000 dilution).

Techniques: Inhibition, Software, Incubation, Blocking Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: IGF1R Is a Potential New Therapeutic Target for HGNET-BCOR Brain Tumor Patients

doi: 10.3390/ijms20123027

Figure Lengend Snippet: Ceritinib acts via the IGF1R/AKT pathway: PhKh1 cells were stimulated after starvation with IGF2 in the presence or absence of ceritinib or vehicle (DMSO). The expression of IGF1R, AKT, ERK1/ERK2, and their respective phosphorylated forms (P-IGF1R, P-AKT, P-ERK1/P-ERK2) was analyzed by western blot with specific antibodies. GAPDH was used as loading control. A representative experiment of three independent experiments is shown.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (MA, USA): GAPDH (14C10) (cat # 2118, 1:1000 dilution), Lamin B1 (D4Q4Z) (cat# 12586, 1:1000 dilution) (Cell Signaling Technology), IGF1 Receptor β (D23H3) XP® (cat # 9750, 1:1000 dilution), Phospho-IGF1 Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) (cat # 3021, 1:1000 dilution), AKT (cat # 9272, 1:1000 dilution), Phospho-AKT (Ser473) (cat # 9271, 1:1000 dilution).

Techniques: Expressing, Western Blot

Journal: Journal of Neuroscience

Article Title: Disturbed Cross Talk between Insulin-Like Growth Factor I and AMP-Activated Protein Kinase as a Possible Cause of Vascular Dysfunction in the Amyloid Precursor Protein/Presenilin 2 Mouse Model of Alzheimer's Disease

doi: 10.1523/jneurosci.4345-06.2007

Figure Lengend Snippet: Figure 3. Actions of AMPK and IGF-I on brain endothelial cells. A, Hypoglycemia increases levelsofpAMPK(theactiveformofthekinase).Brainendothelialcellswerekeptunder0or6.5 mMglucose(euglycemia)for24h.TotallevelsofAMPKwerenotaffectedbytreatments.B,DN AMPK abrogates stimulation of VEGF in endothelial cells after hypoxia (Hyp). Cells were trans- fectedand,after2d,submittedto24hofhypoxia.VEGFlevelsweredetermined1dlater.Cells were lysed and immunoblotted with anti-VEGF, and protein load was determined with anti- PI3K.C,AdditionofAICAR(1mM)totheculturesincreasespAMPKlevels,whereaslevelsofPI3K remainedunchanged.D,Left,StimulationofAMPKincreaseslevelsofVEGFinbrainendothelial cells.CellsweretreatedwithAICAR(topblots)ortransfectedwithaCAAMPK(middleblots)or a DN AMPK (bottom blots) for 2 d. Twenty-four hours later, the cells were lysed and immuno- blotted with anti-VEGF. Right, In the histograms, densitometric quantification of VEGF levels after AICAR or CA AMPK showed significant increases over baseline (*p 0.005; n 4). Representative blots are shown. E, IGF-I and AICAR increased endothelial cell numbers when given separately; however, when given together, no increases were found. *p 0.001 and **p 0.05 vs control (n 4). F, Convergent stimulation of AMPK with AICAR and the IGF-IR with IGF-I results in significantly greater increases in glucose uptake in endothelial cells. Cul- turesreceivedtherespectivetreatments,andglucoseusewasmonitored24hlater.*p0.001 versus control; **p 0.001 versus all other groups (n 4).

Article Snippet: Anti-vascular endothelial growth factor (VEGF) (Oncogene, Cambridge, MA); anti-peroxisome proliferator activator receptor coactivator-1 (PGC-1 ), anti-IGF-I receptor (IGF-IR), and anti-insulin receptor (all from Santa Cruz Biotechnology, Santa Cruz, CA); anti-phosphoThr 172 -AMPK, anti- AMPK, anti-posphoMAPK42– 44 (pMAPK42– 44), and anti-phosphoAkt (pAkt) (all from Cell Signaling Technology, Danvers, MA) were used at dilutions of 1:200 and 1:1000.

Techniques: Control

Journal: Journal of Neuroscience

Article Title: Disturbed Cross Talk between Insulin-Like Growth Factor I and AMP-Activated Protein Kinase as a Possible Cause of Vascular Dysfunction in the Amyloid Precursor Protein/Presenilin 2 Mouse Model of Alzheimer's Disease

doi: 10.1523/jneurosci.4345-06.2007

Figure Lengend Snippet: Figure 4. Interactions between IGF-I and AMPK signaling in brain endothelial cells. A, Costimulation of IGF-IR with IGF-I and AMPK with AICAR inhibits VEGF stimulation. Endothelial cells were exposed for 24 h with the stimuli and processed thereafter. Although IGF-I or AICAR alone produced a significant increase in VEGF levels (top), when both were given together, no increases in VEGF were appreciated. B, Both Akt and MAPK become phosphorylated after exposure of endothelial cells to IGF-I or AICAR; however, when both stimuli are given together, only pAkt levels increase. Brain endothelial cells were stimulated with IGF-I, AICAR,orbothandafter20minlysedandimmunoblottedwithanti-phospho-specificAktSer 473oranti-phospho-specificp44/42 MAPK Thr 202/Tyr 204. Protein load was monitored with anti-PI3K. A representative blot is shown. Time-course analysis (bottom panels) indicated that coaddition of IGF-I and AICAR blunted pMAPK without altering pAkt responses, which, in fact, were prolonged.n4;*p0.001vsrespectivetimeaftereitherIGF-IorAICARalone.C,StimulationofMAPKisrequiredtostimulate VEGFinendothelialcellsafterIGF-I.AdditionoftheMAPKinhibitorPD98059(PD;20M),beforestimulationwith10 7 MIGF-I, inhibits production of VEGF by endothelial cells detected 24 h later (p 0.05 vs control; n 4). A representative blot is shown. Proteinloadwasstandarizedwithanti-PI3K.D,StimulationofVEGFwithAICARwasabrogatedbyinhibitionofeitherMAPKwith PD98059 or PI3K with LY294002 (LY; 25 M; p 0.001 vs control; n 4). E, When cells are transfected with CA AMPK and simultaneously stimulated with IGF-I, MAPK is not phosphorylated. This inhibition is abrogated by cotransfection of endothelial cellswithDNAkt(n2).F,CostimulationofAMPKandtheIGF-IRresultsinAkt-dependentinhibitionofVEGFproduction:aDN Akt recovers VEGF stimulation after IGF-I plus AICAR (AI). Cells were transfected with the respective DNAs and submitted to treatments 2 d later. Twenty minutes and 24 h later, pMAPK and VEGF levels, respectively, were determined. Controls were transfectedwithemptyvectors.Representativeblotsareshownatthetop,anddensitometrichistogramsareshownatthebottom (*p 0.05 vs control; n 4–5). G, Conjoint activation of AMPK and IGF-IR in brain endothelial cells originates a significantly greater resilience to cytotoxic doses of H2O2. Cells were exposed to 100 M H2O2, and 3 h later, cell death was monitored by counting in a fluorescent microscope the number of dead (propidium iodide-labeled) and alive (fluorescein diacetate-positive) cells. *p 0.05 versus control; **p 0.01 versus all other groups (n 5).

Article Snippet: Anti-vascular endothelial growth factor (VEGF) (Oncogene, Cambridge, MA); anti-peroxisome proliferator activator receptor coactivator-1 (PGC-1 ), anti-IGF-I receptor (IGF-IR), and anti-insulin receptor (all from Santa Cruz Biotechnology, Santa Cruz, CA); anti-phosphoThr 172 -AMPK, anti- AMPK, anti-posphoMAPK42– 44 (pMAPK42– 44), and anti-phosphoAkt (pAkt) (all from Cell Signaling Technology, Danvers, MA) were used at dilutions of 1:200 and 1:1000.

Techniques: Produced, Control, Transfection, Inhibition, Cotransfection, Activation Assay, Microscopy, Labeling

Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A . VEGF concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, Fibro+Myo, cCSC4A+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . TGF-β1 concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. all. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. C . IGF-1 concentration following 21 days of cardiomyocyte culture alone (Myo alone) or co-cultured with fibroblasts (Fibro+Myo), GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) or GATA-4 high c-kit pos cCSCs (cCSC4A+Myo). *P<0.05 vs. Myo alone, †P<0.05 vs. Fibro+Myo, cCSC1A+Myo, cCSC3C+Myo. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. D. IGF-1 concentration correlated with cardiomyocyte contractility. Correlation was determined using Pearson Product Moment Correlation coefficient.

Article Snippet: To inhibit the IGF-1 signaling pathway, cardiomyocytes co-cultured with c-kit pos GATA-4 high cCSC4A using inserts for 21 days were harvested following 48 hours treatment with IGF-1 receptor blocking antibody (1 µg/ml; Abcam) or Akt inhibitor (124005, 10 µmol/l; CalBiochem) added to the culture medium.

Techniques: Concentration Assay, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A–C . Representative confocal microscopy images showing GFP pos (green) GATA-4 high c-kit pos cCSCs (cCSC4A+Myo) differentiated into the cardiomyocyte lineage (α-sarcomeric actin pos ; red) when co-cultured with adult rat cardiomyocytes (α-sarcomeric actin pos ; red) over 14 days (B). Note the lack of cardiomyogenic differentiation of GFP pos (green) GATA-4 high c-kit pos cCSCs at 3 days (cCSC4A+Myo; A) and GFP pos (green) GATA-4 low c-kit pos cCSCs at 14 days (cCSC3C+Myo; C). Bar = 20 µm. D . The percent number of GFP pos α-sarcomeric actin pos cells at 3, 7 and 14 days in vitro (DIV) following co-culture of GFP pos GATA-4 low expressing c-kit pos cCSCs with cardiomyocytes (cCSC3C+Myo), GFP pos GATA-4 high c-kit pos cCSCs with cardiomyocytes (cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. cCSC3C+Myo. ** vs. cCSC3C alone. † vs. cCSC4A alone. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. E . The percent number of GFP pos α-sarcomeric actin pos cCSC-derived cells following supplementation with IGF-1 for 14 days in either co-culture conditions (cCSC3C+Myo; cCSC4A+Myo) or culture alone (cCSC3C alone; cCSC4A alone). *P<0.05 vs. without (-) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using t test.

Article Snippet: To inhibit the IGF-1 signaling pathway, cardiomyocytes co-cultured with c-kit pos GATA-4 high cCSC4A using inserts for 21 days were harvested following 48 hours treatment with IGF-1 receptor blocking antibody (1 µg/ml; Abcam) or Akt inhibitor (124005, 10 µmol/l; CalBiochem) added to the culture medium.

Techniques: Confocal Microscopy, Cell Culture, In Vitro, Co-Culture Assay, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A . ELISA assay assessed IGF-1 concentration in the culture medium, with and without FBS, of c-kit pos GATA-4 low cCSC1A, cCSC3C and c-kit pos GATA-4 high cCSC4A, when cultured alone. *P<0.05 vs. cCSC1A, †P<0.05 vs. cCSC3C. Data are Mean ± SD of 3 assays (with 3 triplicates/assay) and analysed using ANOVA. B . RT-PCR analysis showed c-kit pos GATA-4 high cCSC4A had low IGF-1 message levels, compared to rat fibroblasts (Fibro), skeletal muscle cells (SkM) and cardiomyocytes (CaM). C . RT-PCR analysis showed that IGF-1 gene expression level in fibroblasts (Fibro) and c-kit pos GATA-4 high cCSC4A (cCSC4A) is not altered through 7 days of culture in conditioned medium from adult cardiomyocytes (CM (CaM)). Also, IGF-1 gene expression level in cultured cardiomyocytes (CaM) is not altered through culture for 7 days with c-kit pos GATA-4 high cCSC4A conditioned medium (CM (cCSC4A)). D–E . c-kit pos GATA-4 high cCSC4A/cardiomyocyte co-culture conditioned medium, which is high in IGF-1 concentration, increased the percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (-) cCSC4A co-culture conditioned medium. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. F–H . Supplementation of IGF-1 to the culture medium attenuated percent number of TdT-positive cardiomyocytes, increased percent number of beating cardiomyocytes and number of attached cTnI positive cells when cardiomyocytes were cultured alone (Myo Alone), or co-cultured with either fibroblasts (Fibro+Myo), or GATA-4 low expressing c-kit pos cCSCs (cCSC1A+Myo, cCSC3C+Myo) for 7 days. *P<0.05 vs. without (−) IGF-1. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA.

Article Snippet: To inhibit the IGF-1 signaling pathway, cardiomyocytes co-cultured with c-kit pos GATA-4 high cCSC4A using inserts for 21 days were harvested following 48 hours treatment with IGF-1 receptor blocking antibody (1 µg/ml; Abcam) or Akt inhibitor (124005, 10 µmol/l; CalBiochem) added to the culture medium.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Co-Culture Assay

Journal: PLoS ONE

Article Title: c-kit pos GATA-4 High Rat Cardiac Stem Cells Foster Adult Cardiomyocyte Survival through IGF-1 Paracrine Signalling

doi: 10.1371/journal.pone.0014297

Figure Lengend Snippet: A. Representative Western blots show activation of the IGF-1 receptor (IGF-1-R) and downstream signaling to its physiological target, Akt, in cardiomyocytes co-cultured using inserts with c-kit pos GATA-4 high cCSC4A (cCSC4A+Myo), but not in cardiomyocytes cultured alone (Myo Alone) or when co-cultured with c-kit pos GATA-4 low cCSCs (cCSC3C+Myo) for 21 days. Addition of an IGF-1 receptor blocking antibody to the co-culture of c-kit pos GATA-4 high cCSC4A plus cardiomyocytes obliterates IGF-1 signalling in the co-cultured cardiomyocytes. B–C . Optical Density (O.D.) of phospho-IGF-1R (B) and phospho-Akt (C). *P<0.05 vs. cCSC4A+Myo. †P<0.05 vs. cCSC3C+Myo in B. †P<0.05 vs. cCSC3C+Myo, cCSC4A+Myo+IGF-1R Ab in C. Data are Mean ± SD of 3 assays and analysed using ANOVA. D . Representative Western blots shows prevention of Akt phosphorylation in cardiomyocytes when the Akt inhibitor, 124005 was added to the cardiomyocyte/c-kit pos GATA-4 high cCSC4A co-culture. E–F . Number of attached and percent number of beating cardiomyocytes decreased following inhibition of the IGF-1 signaling pathway through treatment with IGF-1 receptor blocking antibody or the Akt inhibitor, at day 19 for 48 hours. Data are Mean ± SD for 3 wells/condition and analysed using ANOVA. *P<0.05 vs. cCSC4A+Myo.

Article Snippet: To inhibit the IGF-1 signaling pathway, cardiomyocytes co-cultured with c-kit pos GATA-4 high cCSC4A using inserts for 21 days were harvested following 48 hours treatment with IGF-1 receptor blocking antibody (1 µg/ml; Abcam) or Akt inhibitor (124005, 10 µmol/l; CalBiochem) added to the culture medium.

Techniques: Western Blot, Activation Assay, Cell Culture, Blocking Assay, Co-Culture Assay, Inhibition

Journal: Journal of cell science

Article Title: Growth arrest induces primary-cilium formation and sensitizes IGF-1-receptor signaling during differentiation induction of 3T3-L1 preadipocytes.

doi: 10.1242/jcs.046276

Figure Lengend Snippet: Fig. 3. IGF-1-receptor activation in ciliated 3T3-L1 preadipocytes. (A) IGF-1- receptor activation. Proliferating or 2-day post-confluent 3T3-L1 preadipocytes were treated with 100 nM insulin for the indicated time. The activated receptor was western blotted for Tyr1131 phosphorylation (PY-IGF- 1R). (B) Immunofluorescence staining of IGF-1 receptor and primary cilium. 2-day post-confluent 3T3-L1 preadipocytes treated with 100 nM insulin for 1 minute or not (control) were stained for acetylated α-tubulin (Ac-Tub) and IGF-1-receptor β-subunit (IGF-1R). Arrowheads indicate the primary cilium. (C) Immunofluorescence staining of activated IGF-1 receptor. A 2-day post- confluent preadipocyte was treated with 10 nM or 100 mM insulin for 1 minute, or untreated (control), and stained for acetylated α-tubulin and Tyr1131-phosphorylated IGF-1 receptor β-subunit. Arrowheads indicate the primary cilium. (D) Enlarged image of primary cilium and Tyr1131- phosphorylated IGF-1 receptor. Scale bars: 10 μm.

Article Snippet: Anti-phospho-IGF-1 receptor (Tyr1131) antibody was from Cell Signaling.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Immunofluorescence, Staining, Control

Journal: Journal of cell science

Article Title: Growth arrest induces primary-cilium formation and sensitizes IGF-1-receptor signaling during differentiation induction of 3T3-L1 preadipocytes.

doi: 10.1242/jcs.046276

Figure Lengend Snippet: Fig. 6. Inhibition of IGF-1-receptor signaling in IFT88-knockdown preadipocytes. (A) Inhibition of the IGF-1 receptor and Akt-1 activation in IFT88-knockdown cells. 2-day post-confluent IFT88-knockdown preadipocytes (IFT88 siRNA) or control virus-infected preadipocytes (control siRNA) were treated with 100 nM insulin for the indicated time and harvested for western blot of Tyr1131-phosphorylated IGF-1-receptor β-subunit (PY-IGF-1R) and Ser473-phosphorylated Akt-1 (pAkt-1). (B) Inhibition of Akt-1 activation at the basal body by IFT88 knockdown. The arrowheads indicate the basal body. 2-day post-confluent cells were treated with 100 nM insulin for 1 minute (+Ins) or not (–Ins) and stained for primary cilium (Ac-Tub) and Ser473-phosphorylated Akt-1 (pAkt). IFT88 Ri, IFT88- knockdown preadipocyte; Ctrl Ri, control virus-infected preadipocyte. Scale bar: 10 μm. (C) Rescue of IFT88 knockdown by FLAG-tagged human IFT88. C, control-RNAi virus-infected preadipocyte; Si, preadipocyte infected with IFT88 RNAi virus (S3) and blank expressing virus; R, preadipocyte infected with IFT88 RNAi virus and virus expressing FLAG-tagged human IFT88. (D) Adipocyte differentiation. Standard differentiation protocol was followed and cells were stained with Oil-red-O on day 8. (E) Expression of C/EBPα and PPARγ in rescued cells. Numbers (0, 2, 4, 6, 8) indicate the days after differentiation induction. IFT88 siRNA, cell infected with IFT88 RNAi virus (S3) and blank expressing virus; siRNA rescue, cell infected with IFT88 RNAi virus and virus expressing FLAG- tagged human IFT88. (F) The activation of IGF-1 receptor and Akt-1 in rescued cells. The labels are the same as in panel A.

Article Snippet: Anti-phospho-IGF-1 receptor (Tyr1131) antibody was from Cell Signaling.

Techniques: Inhibition, Knockdown, Activation Assay, Control, Virus, Infection, Western Blot, Staining, Expressing

Journal: Journal of cell science

Article Title: Growth arrest induces primary-cilium formation and sensitizes IGF-1-receptor signaling during differentiation induction of 3T3-L1 preadipocytes.

doi: 10.1242/jcs.046276

Figure Lengend Snippet: Fig. 8. Inhibition of IGF-1-receptor signaling in Kif3a-knockdown preadipocytes. (A) Primary-cilium formation is blocked by Kif3a knockdown. ctrl siRNA, preadipocyte infected with control virus; Kif3a siRNA, preadipocyte infected with Kif3a RNAi virus (S3). Scale bars: 10 μm. (B) Kif3a expression. 2-day post- confluent 3T3-L1 preadipocytes infected with Kif3a RNAi retrovirus (S1, S2 or S3) or control virus (C) were analyzed for Kif3a expression. S3 retrovirus was used in the subsequent experiment. (C) Adipocyte differentiation. Standard differentiation protocol was followed and cells were stained with Oil-red-O on day 8. (D) Expression of adipocyte transcription factors. The numbers (0, 2, 4, 6, 8) are the days after differentiation induction. C/EBPα and PPARγ were analyzed by western blot. (E) Inhibition of IGF-1 receptor and Akt-1 activation in Kif3a-knockdown cells. 2-day post-confluent Kif3a-knockdown preadipocytes (Kif3a siRNA) or control virus-infected preadipocytes (control siRNA) were treated with 100 nM insulin for indicated time and harvested for western blot of Tyr1131- phosphorylated IGF-1-receptor β-subunit (PY-IGF-1R) and Ser473-phosphorylated Akt-1 (pAkt-1).

Article Snippet: Anti-phospho-IGF-1 receptor (Tyr1131) antibody was from Cell Signaling.

Techniques: Inhibition, Knockdown, Infection, Control, Virus, Expressing, Staining, Western Blot, Activation Assay