Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling

doi: 10.1186/s12964-018-0273-7

Figure Lengend Snippet: IGFBP6 and IGF2 are differentially secreted by TMZ-sensitive and TMZ-resistant glioma cells. a Determination of IGF2 and IGFBP6 expression in conditioned medium of TMZ-sensitive (U251, left) and TMZ-resistant (UTMZ, right) glioma cells using a human growth factor antibody array. b Quantitative analysis of IGF2 (left) and IGFBP6 (right) levels in culture medium of U251 and UTMZ glioma cells determined by ELISA. c Relative expression of IGF2 (left) and IGFBP6 (right) mRNA in tumor samples from GBM patients with OS less than or greater than 6 months. d Relative expression of IGF2 (left) and IGFBP6 (right) mRNA in TMZ-sensitive and TMZ-resistant GBM xenograft lines. mRNA expression was determined by qRT-PCR and normalized to 18S. * P < 0.05, ** P < 0.001, **** P < 0.0001. Two-tailed, t-test

Article Snippet: The membranes were blocked for 1 h and incubated overnight at 4 °C with the following primary antibodies: AKT (5G3) Mouse mAb #2966, IGF-I Receptor β (D23H3) XP Rabbit mAb #9750, phospho-IGF1 Receptor β (Tyr1135/1136) /Insulin Receptor β (Tyr1150/1151) (19H7) Rabbit mAb #3024, phospho-AKT (Ser473) (D9E) XP Rabbit mAb #4060, and Rab11 (D4F5) XP Rabbit mAb #5589 (all from Cell signaling at a dilution of 1:1000), and IGFBP6 antibody (C-20) (Santa Cruz, #sc-6007, dilution 1:500).

Techniques: Expressing, Ab Array, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling

doi: 10.1186/s12964-018-0273-7

Figure Lengend Snippet: Activation of IGF-1R is abrogated after in vitro treatment with IGFBP6. a TMZ-sensitive (U251) and TMZ-resistant (UTMZ) cells were cultured in serum-free medium (SFM) for 16 h, then stimulated by IGF2 (50 ng/ml) for the indicated time. Cell lysates were subjected to Western blot analysis to determine the phosphorylation of IGF-1R and AKT (top panel) and total IGF-1R and AKT (bottom panel) in TMZ-resistant cells. AKT was constitutively activated in TMZ-sensitive cells. Representative blots are shown. b Quantitative analysis of protein expression levels from blots shown in ( a ). Bars represent the average from triplicate determinations from at least three independent experiments. c TMZ-resistant cells were starved in SFM overnight and then stimulated with IGF2 (50 ng/ml) in the presence or absence of recombinant IGFBP6 (100 or 200 ng/ml). Representative Western blots showing that IGFBP6 (but not IGFBP2) abrogated the IGF2-dependent phosphorylation of IGF-1R and AKT. Expression of Rab11 is shown as the loading control

Article Snippet: The membranes were blocked for 1 h and incubated overnight at 4 °C with the following primary antibodies: AKT (5G3) Mouse mAb #2966, IGF-I Receptor β (D23H3) XP Rabbit mAb #9750, phospho-IGF1 Receptor β (Tyr1135/1136) /Insulin Receptor β (Tyr1150/1151) (19H7) Rabbit mAb #3024, phospho-AKT (Ser473) (D9E) XP Rabbit mAb #4060, and Rab11 (D4F5) XP Rabbit mAb #5589 (all from Cell signaling at a dilution of 1:1000), and IGFBP6 antibody (C-20) (Santa Cruz, #sc-6007, dilution 1:500).

Techniques: Activation Assay, In Vitro, Cell Culture, Western Blot, Phospho-proteomics, Expressing, Recombinant, Control

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling

doi: 10.1186/s12964-018-0273-7

Figure Lengend Snippet: Abrogation of IGF2-mediated phosphorylation of IGF-1R is mediated by IGFBP6 binding and sequestration of IGF2. a Schematic representation of human IGFBP6 (Swiss-Prot, P24592) showing mutated sites. Amino acids mutated to Ala are shown in red. b Western blot analysis of recombinant IGFBP6 proteins (top panel). wt, wt-IGFBP6; mut, mut-IGFBP6; wt c , commercial wt-IGFBP6. Western blot ligand binding analysis (bottom panel). Wt-IGFBP6 (250 ng) and mut-IGFBP6 (250 ng or 1 μg) proteins were separated on 4–20% gels and transferred to PVDF membranes, incubated with Strep-Tag II-IGF2 and then developed with NWSHPQFEK Tag antibody. c Representative Western blot showing that wt-IGFBP6 (but not mut-IGFBP6) abrogates the IGF2-dependent phosphorylation of IGF-1R and AKT in TMZ-resistant cells. UTMZ cells were cultured in SFM for 16 h and then stimulated with IGF2 (50 ng/ml) in the presence or absence of recombinant wt-IGFBP6 or mut-IGFBP6 (200 ng/ml). d Representative Western blot showing that conditioned medium from chemosensitive cells abrogates the IGF2-dependent phosphorylation of IGF-1R and AKT in chemoresistant cells. UTMZ cells were cultured in SFM overnight and then stimulated with IGF2 at the indicated doses in the presence of conditioned medium from U251 cells. Expression of Rab11 is shown as the loading control. e Quantitative analysis (top) of extracellular IGFBP6 in conditioned medium of UTMZ cells stably transfected with one of five IGFBP6-shRNA constructs or a non-targeted control shRNA construct. Extracellular IGFBP6 was detected by Western blot (bottom) after 48 h of culture in SFM. Data are presented as the mean ± SEM of three independent experiments (* P < 0.05, ** P < 0.001). f Representative Western blot showing that abrogation of the IGF2-dependent phosphorylation events requires the binding and sequestration of IGF2 by IGFBP6. UTMZ cells were cultured in SFM overnight and then stimulated with IGF2 at the indicated doses in the presence of conditioned medium from TMZ-sensitive U251 cells transfected with control shRNA or IGFBP6-shRNA constructs. Expression of Rab11 is shown as the loading control. CM, conditioned medium; CS, TMZ-sensitive cells; NT, non-targeted

Article Snippet: The membranes were blocked for 1 h and incubated overnight at 4 °C with the following primary antibodies: AKT (5G3) Mouse mAb #2966, IGF-I Receptor β (D23H3) XP Rabbit mAb #9750, phospho-IGF1 Receptor β (Tyr1135/1136) /Insulin Receptor β (Tyr1150/1151) (19H7) Rabbit mAb #3024, phospho-AKT (Ser473) (D9E) XP Rabbit mAb #4060, and Rab11 (D4F5) XP Rabbit mAb #5589 (all from Cell signaling at a dilution of 1:1000), and IGFBP6 antibody (C-20) (Santa Cruz, #sc-6007, dilution 1:500).

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Recombinant, Ligand Binding Assay, Incubation, Strep-tag, Cell Culture, Expressing, Control, Stable Transfection, Transfection, shRNA, Construct

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling

doi: 10.1186/s12964-018-0273-7

Figure Lengend Snippet: IGFBP6 abrogates proliferation of TMZ-resistant cells in vitro and in vivo. a GFP-labelled TMZ-resistant UTMZ cells were co-cultured with unlabeled TMZ-sensitive U251 cells transfected with non-targeted shRNA (control) or IGFBP6-shRNA (20%:80% ratio). After 48 h of co-culture the percentage of each cell population was determined by flow cytometry. Left: representative histograms show the distribution of GFP + and GFP − cells. Right: the percentage of GFP − cells (top graph) and GFP + cells (bottom graph) quantified after 48 h of co-culture. Data are presented as the mean ± SEM of three independent experiments (* P < 0.05, ** P < 0.001). b UTMZ cells were plated at equal densities in 96-well plates and exposed to the indicated doses of wt-IGFBP6 or mut-IGFBP6. After 96 h, cell number was measured by CyQUANT Cell Proliferation Assays Kit. Results represent the mean of four independent experiments, with each treatment performed in quadruplicate within each experiment. (* P < 0.05, ** P < 0.001, **** P < 0.0001). c UTMZ cells were transfected to stably overexpress IGFBP6. The representative Western blot shows the expression of IGFBP6 protein in the extracellular medium after 48 h of culture in SFM (top panel). Empty vector-transfected or IGFBP6-transfected UTMZ cells (1 × 10 5 ) were intracranially implanted in athymic nude mice ( n = 5). A significant improvement in survival was observed in mice bearing IGFBP6-transfected cells (Log-rank test, ** P = 0.0018). Numbers in parentheses indicate median survival. d Representative images of tumors from athymic nude mice inoculated in the flank with empty vector-transfected or IGFBP6-transfected UTMZ cells (0.5 × 10 6 ). Tumors were excised 50 days after inoculation (left panel). Comparison of tumor weights upon excision (right panel). (** P < 0.001). CR, TMZ-resistant; NT, non-targeted

Article Snippet: The membranes were blocked for 1 h and incubated overnight at 4 °C with the following primary antibodies: AKT (5G3) Mouse mAb #2966, IGF-I Receptor β (D23H3) XP Rabbit mAb #9750, phospho-IGF1 Receptor β (Tyr1135/1136) /Insulin Receptor β (Tyr1150/1151) (19H7) Rabbit mAb #3024, phospho-AKT (Ser473) (D9E) XP Rabbit mAb #4060, and Rab11 (D4F5) XP Rabbit mAb #5589 (all from Cell signaling at a dilution of 1:1000), and IGFBP6 antibody (C-20) (Santa Cruz, #sc-6007, dilution 1:500).

Techniques: In Vitro, In Vivo, Cell Culture, Transfection, shRNA, Control, Co-Culture Assay, Flow Cytometry, CyQUANT Assay, Stable Transfection, Western Blot, Expressing, Plasmid Preparation, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling

doi: 10.1186/s12964-018-0273-7

Figure Lengend Snippet: Model of paracrine-mediated regulation of chemoresistant cell proliferation in the context of glioma tumor heterogeneity. a TMZ-sensitive cells (blue) secrete IGFBP6, which binds and sequesters IGF2 produced by TMZ-resistant cells (red). As a consequence, IGF-1R and AKT signaling is not activated by IGF2, and proliferation of TMZ-resistant cells is suppressed. After TMZ treatment ( b and c ), the population of IGFBP6-producing, TMZ-sensitive cells is reduced, leaving extracellular IGF2 free and thus able to bind to and activate IGF-1R on the TMZ-resistant cells, thereby activating AKT signaling. Coordinate activation of IGF-1R and AKT enhances the proliferation of these chemoresistant glioma cells

Article Snippet: The membranes were blocked for 1 h and incubated overnight at 4 °C with the following primary antibodies: AKT (5G3) Mouse mAb #2966, IGF-I Receptor β (D23H3) XP Rabbit mAb #9750, phospho-IGF1 Receptor β (Tyr1135/1136) /Insulin Receptor β (Tyr1150/1151) (19H7) Rabbit mAb #3024, phospho-AKT (Ser473) (D9E) XP Rabbit mAb #4060, and Rab11 (D4F5) XP Rabbit mAb #5589 (all from Cell signaling at a dilution of 1:1000), and IGFBP6 antibody (C-20) (Santa Cruz, #sc-6007, dilution 1:500).

Techniques: Produced, Activation Assay

Journal: Oncotarget

Article Title: Human monocyte-derived dendritic cells exposed to hyperthermia show a distinct gene expression profile and selective upregulation of IGFBP6

doi: 10.18632/oncotarget.18338

Figure Lengend Snippet: A ., B. The dot plots, obtained by plotting the area of the cells (x-axis) vs the aspect ratio ( i.e. the ratio between length and height) parameter (y-axis), displaying the typical distribution of DCs in presence (A) or in absence (B) of permeabilization, show that permeabilization did not alter cell viability. C. , D. , E. , F. The percentage of cells expressing IGFBP-6 after exposure at 39°C at different times and the relative percentage of mean fluorescence intensity (MFI) of cells exposed at 39°C towards cells exposed at 37°C (considered as 100%) were analyzed by flow cytometry in the presence (C and D, respectively) or absence of permeabilization (E and F, respectively). No staining was seen with controls using either secondary antibody alone or isotype control rabbit IgG polyclonal antiserum (not shown). Each bar represents mean ±SEM of four experiments. * p < 0.05; ** p < 0.001. Statistical comparisons were made using unpaired Student’s t-test.

Article Snippet: In order to assess for the specificity of the IGFBP-6 chemotactic effect, we pre-incubated IGFBP-6 at the highest dose with a rabbit IgG anti-human C-terminal IGFBP-6 antibody (Abcam, Cambridge, UK) or with an irrelevant rabbit IgG antibody (anti-human HVCN1, Abcam, Cambridge, UK) in a weight ratio (IGFBP-6/antibody) of 1:6 for 30 min at room temperature, before adding the mixture in the lower compartment.

Techniques: Expressing, Fluorescence, Flow Cytometry, Staining