anti igfbp6 blocking antibody  (R&D Systems)

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Western blot analysis evaluating the expression of ITGA6 and CD63 (marker of extracellular vesicles) in conditioned medium (CM) and whole cell lysates of TOV-112D PT-res cells treated with CDDP for the indicated time points. ( B ) Western blot analysis to compare ITGA6 and CD63 expression in whole lysates (LYS), CM, exosomes (EXO) and exosomes-depleted CM (DEPR). ( C ) Western blot analysis of ITGA6 and CD63 in exosomes isolated from CM of the indicated cells treated with CDDP for 3, 6, and 9 h. ( D , E ) Graphs reporting the area of ovaryspheres formed by TOV-112D parental cells primed with exosomes ( D ) or CM ( E ) of TOV-112D PT-res ITGA6WT and KO cells and then plated on a mesothelial cells monolayer (NC = Not Conditioned). In ( E ), representative phase-contrast images were also reported under the graph. Scale bars, 50 μm. In ( D ) and ( E ), data represent the median (± SD) of three independent experiments performed in triplicate in which at least 70 randomly selected cells were analyzed. Statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). ( F ) Western blot analysis evaluating the expression of the indicated proteins in whole cell lysates of TOV-112D PT-sen cells challenged for the indicated time points with the CM from TOV-112D PT-res ITGA6WT or ITGA6KO cells. NC = Not Conditioned. ( G ) Western blot analysis evaluating the expression of the indicated proteins in TOV-112D PT-sen cells incubated or not (NC) for the indicated times with the CM from TOV-112D PT-res WT in the presence or not of the anti-ITGA6 blocking antibody, GoH3. ( H ) Western blot analysis evaluating the expression of the indicated proteins in TOV-112D PT-sen cells incubated or not (NC) for the indicated times with the CM from TOV-112D PT-res WT cells in the presence or not of the Src inhibitor, Saracatinib. ( I ) Co-immunoprecipitation (Co-IP) analysis of ITGA6 and IGF1Rβ receptor in TOV-112D PT-res ITGA6WT or ITGA6KO cells. ( J ) Co-IP analysis of ITGA6 and IGF1Rβ receptor in TOV-112D parental cells incubated or not (NC) with the CM from TOV-112D PT-res WT in the presence or not of the anti-ITGA6 blocking antibody, GoH3. In ( H ) and ( I ), input shows the expression of the indicated proteins in the lysates used for the IP experiments; IgG represents the control IP using an unrelated antibody. ( K ) Table reporting the expression of the 7 proteins related to IGF1R pathway present in the used cytokine array. The mean fold ( n = 3 replicates) reports the ratio between the expression of each cytokine in the CM of PT-res ITGA6KO compared to that in the CM of PT-res ITGA6WT cells. Statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). ( L ) Western blot analysis evaluating the expression of IGFBP6 in CM and whole lysates of TOV-112D PT-res ITGA6WT or ITGA6KO cells. ( M ) Western blot analysis of the indicated proteins in cell lysates of TOV-112D PT-sen cells incubated or not (NC) for the indicated times with the CM of TOV-112D PT-res ITGA6 KO cells in presence or not of the specific anti-IGFBP6 blocking antibody. In the figure GAPDH was used as loading control. .

Article Snippet: In both functional assays, the challenging of TOV-112D PT-sen cells or mesothelial cells with CM from ITGA6 WT or KO cells, could be also in presence or absence of anti-ITGA6 blocking antibody, GoH3 (10 μg/ml) or anti-IGFBP6 blocking antibody (15 μg/ml) (AF876, R&D System) as indicated.

Techniques: Western Blot, Expressing, Marker, Isolation, Two Tailed Test, Incubation, Blocking Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Control

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Schematic representation of the experimental procedures used. Mesothelial cells (LPL) cells were incubated or not with CM of TOV-112D PT-res ITGA6WT or ITGA6KO cells for 16 h and then used for adhesion assays with TOV-112D PT-sen cells or for protein and mRNA expression analysis by western blot and qRT-PCR. NC = Not Conditioned. ( B ) Graph (left) and representative phase-contrast images (right) reporting the area of ovaryspheres formed by TOV-112D PT-sen cells plated on mesothelial cells monolayer primed as described in ( A ). Data represent the median (± SD) of three independent experiments performed in triplicate in which at least 70 randomly selected cells were analyzed. ( C ) Western blot analysis evaluating the expression of IGFBP6 and IGF2 in mesothelial whole cell lysates treated as described in ( A ). ( D ) Western blot analysis evaluating the expression of the indicated proteins in whole cell lysates of mesothelial cells conditioned or not (NC) for the indicated times with CM of TOV-112D PT-res ITGA6KO cells in presence or not of the specific anti-ITGA6 GoH3 Ab. In ( C ) and ( D ), Tubulin was used as loading control. ( E ) Graph reporting the mRNA expression of IGF2 and LAMA5 (LM) in mesothelial cells incubated for the indicated times as described in ( A ). NC = Not Conditioned. Data are the mean (± SD) of 3 replicates. ( F ) Clinical history of patient #1 reporting the timeline of chemotherapy treatments and ascites collections. The amount of CA125 (in blood samples) and ITGA6 (in ascites) were reported in red and blue, respectively. ( G ) Graph (top) and representative phase-contrast images (bottom) reporting the area of ovaryspheres formed by TOV-112D PT-sen cells plated on mesothelial cells monolayer conditioned or not (NC) for 16 h with ascites samples described in ( F ), in the presence of the specific anti-ITGA6 blocking antibody GoH3, as indicated. Data represent the median (± SD) of three independent experiments performed in triplicate in which at least 70 randomly selected cells were analyzed. ( H ) Schematic representation depicting the role of ITGA6 in inducing a PT-resistant phenotype (increased invasion, adhesion, and stemness) both at cell-autonomous and non-cell-autonomous levels, leading to the formation of the pre-metastatic niche. Overexpression of ITGA6 allows a better adhesion of PT-res cells to the mesothelium. ITGA6 engagement induces stabilization of Snail and inhibition of IGFBP6 expression that promote invasion and stem-like behavior. On the other side secreted ITGA6 acts on PT-sen and mesothelial cells to activate the IGF1R pathway and amplify the pro-metastatic signals. ITGA6 KO, or its inhibition with blocking antibodies, prevents the activation of pro-metastatic metastatic pathways both in PT-res cells and in recipient cells and impairs IGF1R signaling by releasing the inhibition on IGFBP6 transcription leading to its overproduction. In ( B ) and ( G ), data represent the mean (± SD) of two independent experiments performed in triplicate in which at least 10 randomly selected fields were analyzed. Scale bars, 50 μm. In the figure statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). .

Article Snippet: In both functional assays, the challenging of TOV-112D PT-sen cells or mesothelial cells with CM from ITGA6 WT or KO cells, could be also in presence or absence of anti-ITGA6 blocking antibody, GoH3 (10 μg/ml) or anti-IGFBP6 blocking antibody (15 μg/ml) (AF876, R&D System) as indicated.

Techniques: Incubation, Expressing, Western Blot, Quantitative RT-PCR, Control, Blocking Assay, Over Expression, Inhibition, Activation Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Schematic representation of the in vivo experimental procedures. NSG mice injected intraperitoneally (IP) with TOV-112D PT-res ITGA6WT ( n = 11) or ITGA6KO ( n = 11) cells, were randomly divided into two group and treated ( n = 6) or not ( n = 5) with CBDCA as indicated. Mice were sacrificed 30 days after the injection. ( B ) Graph reporting total number of tumors in mice described in ( A ), determined by macroscopic and pathological analyses (total tumor burden). ( C , D ) Radar ( C ) and Dot ( D ) plots reporting the distribution of abdominal metastasis ( C ) and the total number of organs infiltrated by cancer cells ( D ) in mice described in ( A ). In ( C ), black (WT cells) and red (ITGA6KO cells) lines indicate the number (values 0 to 5 for untreated and 0 to 6 for CBDCA-treated mice) of mice affected for each district, as indicated. Typical images of hematoxylin and eosin (H&E) staining of the showing liver metastasis in mice injected with ITGA6WT PT-res cells and treated as described in ( A ). 5X (scale bar = 200 μm) and 10X (scale bar = 50 μm) images of the same field are shown. White dashed boxes represent the magnified areas. ( E , G ) Western blot analysis evaluating the expression of ITGA6 and Snail in tumor masses ( E ) and IGFBP6 in the tumor masses ( F ) and ascites ( G ) of mice described in ( A ). Tubulin and Ponceau were used as loading controls. Mice 3 and 4 injected with ITGA6KO cells were treated with CBDCA. ( H ) Schematic representation of the in vivo experimental procedures using EOC PDX model. NSG mice injected IP with PDX OV218.3 ( n = 20), were randomly divided into four groups and treated or not ( n = 5) with the specific anti-ITGA6 blocking antibody ( n = 5), P5G10 ( n = 5), with CBDCA ( n = 5) or with the combination of both ( n = 5), according to the scheme. ( I ) Radar plots reporting the distribution of abdominal metastasis in mice injected intraperitoneally and treated as in ( H ), as determined by macroscopic and pathological analyses. Colored bold lines in each plot indicate the number (values 0 to 5) of mice affected for each district. ( J ) Typical images of H&E analyses of the omentum and lungs of mice described in ( H ). For each condition, 20X (for omentum, scale bar = 50 μm) and 40X (for lungs, scale bar = 20 μm) images are shown. Yellow dashed lanes in lung images highlighted the tumor metastasis. ( K ) Graph reporting total number of metastasis/mouse treated as indicated and determined by pathological analyses ( L ) Western Blot analysis of γH2AX in tumor cells isolated from ascites of mice described in ( H ). ( M ) Typical images of Snail (green) expression on peritoneal metastasis collected from mice described in ( H ) and evaluated by IF analyses (nuclei are in blue). White dashed lines indicate the boundary between tumor masses and the peritoneal wall. Scale bars = 20 μm. In ( B ), ( D ), and ( K ) Statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation. .

Article Snippet: In both functional assays, the challenging of TOV-112D PT-sen cells or mesothelial cells with CM from ITGA6 WT or KO cells, could be also in presence or absence of anti-ITGA6 blocking antibody, GoH3 (10 μg/ml) or anti-IGFBP6 blocking antibody (15 μg/ml) (AF876, R&D System) as indicated.

Techniques: In Vivo, Injection, Staining, Western Blot, Expressing, Blocking Assay, Isolation, Two Tailed Test, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A , B ) Graph reporting the volume of ascitic fluids ( A ) and of explanted macroscopically identified tumors of NSG mice injected intraperitoneally with PT-res ITGA6 WT ( n = 11) and KO cells ( n = 11) and treated ( n = 6) or not ( n = 5) with CBDCA 30 mg/kg 3 times per week for 2 weeks. ( C ) Typical images of mice described in ( A ) and ( B ). Red arrows indicated the presence of macroscopically visible tumors. ( D ) Clinical history reporting the timeline of surgery, chemotherapy treatments (in green) and ascites collection of EOC patient who donate her ascites to establish PDX OV218.3 (see Methods section). ( E , F ) Graph reporting the volume of ascitic fluids ( E ) and number of tumor spheroids ( F ) in NSG mice ( n = 5/group of treatment) injected with PDX OV218.3 and treated or not with the specific anti-ITGA6 blocking antibody P5G10, with CBDCA or with the combination of both according to the scheme reported in Fig. . ( G ) IGFBP6 mRNA expression in tumor cells in ascites of mice described in ( E , F ). mRNA expression was analyzed in triplicate and normalized to actin housekeeping gene expression. The mean (±SD) expression for each mouse is reported in the graph. ( H ) IF analyses evaluating the expression of pIGF1Rβ (green) and ITGA6 (red) on peritoneal metastases collected from mice treated as indicated (nuclei are in blue). White dashed lines indicate the boundary between tumor masses and the peritoneal wall. Yellow dashed boxes represent the areas magnified in the zoomed images, on the right. Scale bars = 20 μm. In the figure, statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation.

Article Snippet: In both functional assays, the challenging of TOV-112D PT-sen cells or mesothelial cells with CM from ITGA6 WT or KO cells, could be also in presence or absence of anti-ITGA6 blocking antibody, GoH3 (10 μg/ml) or anti-IGFBP6 blocking antibody (15 μg/ml) (AF876, R&D System) as indicated.

Techniques: Injection, Blocking Assay, Expressing, Gene Expression, Two Tailed Test, Standard Deviation

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a Volcano plot showing the distribution of DEGs. Yellow dots indicate significantly up-regulated genes, green dots indicate significantly down-regulated genes, and pink dots indicate non-differentially expressed genes. b Heat map of relevant DEGs between fibrosis and control groups. c Top 20 most significantly differentially expressed genes, with IGFBP6 showing the highest significance. d Validation using dataset GSE130123 . CCL4-induced 4 weeks hepatic fibrosis model in mice. (control group n = 4, and fibrosis group n = 5). e IHC staining of COL1A1 and IGFBP6 in hepatic sections from control mice and mice at 4 or 8 weeks after intraperitoneal TAA injection (scale bar: 25 μm). Schematic diagram generated by Biorender (agreement number: AR28HM9B0Q). f Primary HEPs ( n = 6) and HSCs ( n = 6) were isolated from the same liver. The mRNA expression of mouse Igfbp6 . g Primary HSCs were isolated from WT mice treated with three doses of vehicle (PBS, n = 6) or TAA (100 mg/kg, n = 6) every 3 days. Igfbp6 mRNA was measured. h scRNA-seq dataset ( GSE221481 ) from mice with uninjured liver or TAA-induced hepatic fibrosis was analyzed for the expression of Igfbp6 . The fraction of cells expressing Igfbp6 in different cell lineages in liver. Data are expressed as mean ± SD, unpaired Student’s t test, two-tailed.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Control, Biomarker Discovery, Immunohistochemistry, Injection, Generated, Isolation, Expressing, Two Tailed Test

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a qRT-PCR analysis of Acta2 , Fn1 , Col1a1 , and Igfbp6 mRNA levels and b Western blotting analysis of FN, COL1A1, α-SMA, and IGFBP6 protein expression in hepatic tissue homogenates from control and TAA-treated mice at 4 and 8 weeks ( n = 6), Data are expressed as means ± SD, One-way ANOVA. c qRT-PCR analysis of Acta2 , Fn1 , Col1a1 and Igfbp6 mRNA levels in primary mouse HSCs stimulated with TGF-β (10 ng/mL) for 12 h and 24 h or unstimulated ( n = 3), Data are expressed as means ± SD, One-way ANOVA. d Western blotting analysis of protein expression levels of FN, COL1A1, α-SMA and IGFBP6 in primary mouse HSCs stimulated with TGF-β for 24 and 48 h or unstimulated. e –g Western blotting confirmed IGFBP6-knockout in LX2 cells line ( e ). Fibrosis-related gene and protein expression in LX2 cells line stimulated with TGF-β (10 ng/mL) with or without IGFBP6-knockout detected by ( f ) qRT-PCR and ( g ) Western blotting ( n = 3). Data are expressed as means ± SD, Tow-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Knock-Out

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a SwissADME analysis of 30 FDA-approved compounds related to IGFBP6 showing: Heatmap of five drug-likeness rules (Lipinski, Ghose, Veber, Egan, Muegge) and eight pharmacokinetic parameters (GI absorption, BBB permeation, P-gp substrate, CYP1A2/2C19/2C9/2D6/3A4 inhibition). b Bioavailability scores of the compounds. Effect of five candidate compounds on fibrosis-related indicators in TGF-β (10 ng/mL) stimulated primary mouse HSCs were detected ( c ) at 24 h for mRNA levels and ( d ) at 48 h for protein levels ( n = 3). e Chemical structural of PA. f The effect of PA on cell viability of LX2 cells viability was measured using the CCK8 assay ( n = 5). The effect of concentration gradients (0 μM, 0.1 μM, 1 μM, and10μM) of PA against FN, COL1A1, ACTA2, and IGFBP6 in TGF-β (10 ng/mL) -stimulated LX2 cells was examined using ( g ) qRT-PCR for mRNA levels and ( h ) Western blotting for protein levels ( n = 3). Data are expressed as means ± SD, Two-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Inhibition, CCK-8 Assay, Concentration Assay, Quantitative RT-PCR, Western Blot

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a Three-dimensional binding structure of PA (green) with IGFBP6 (gray ribbon). The Pi-H interactions are depicted as magenta dashed lines. b MST analysis of His-tagged IGFBP6 binding to PA ( n = 3). c CETSA melting curves and derived thermal stability profiles ( n = 3). d Western blotting detection of protein expression levels of FN, COL1A1 and α-SMA in IGFBP6-knockout LX2 cells by PA (10 μM) intervention. The effect of PA’s inhibitory action on TGF-β-induced IGFBP6 overexpression in LX2 cells was assessed by Western blotting in the presence or absence of ( e ) the proteasome inhibitor MG132 or ( f ) the autophagy inhibitor 3-MA ( n = 3). Data are expressed as means ± SD, Two-way ANOVA. g LX2 cells were treated with 1 µM PA in the presence or absence of MG132 for 12 h. IP detection was performed with anti-IGFBP6 antibody, and expression of ubiquitin-coupled IGFBP6 was detected with anti-UB antibody ( n = 3). Data are expressed as means ± SD, Two-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Binding Assay, Derivative Assay, Western Blot, Expressing, Knock-Out, Over Expression, Ubiquitin Proteomics

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: a Enrichment analysis results after collection of PA targets via Pharmmapper database and GeneCards database. b Western blotting assay for the influence of PA (10 μM) on different times of TGF-β (10 ng/mL) stimulation-initiated SMAD2/3 phosphorylation levels in primary mouse HSCs ( n = 3). c Effects of different concentrations of PA (0 μM, 0.1 μM, 1 μM, and10 μM) on the phosphorylation level of SMAD2/3 induced by TGF-β (10 ng/mL) action in primary HSCs of mice analyzed by Western blotting ( n = 3). Effect of PA on phosphorylation levels of SMAD2/3 and IGFBP6 in TAA-induced fibrotic hepatic mice in vivo by ( d ) Western blotting and ( e ) IHC ( n = 6, scale bar: 50 μm).

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Western Blot, Phospho-proteomics, In Vivo

Journal: Communications Biology

Article Title: Pantothenic acid ameliorates hepatic fibrosis by targeting IGFBP6 to regulate the TGF-β/SMADs pathway

doi: 10.1038/s42003-025-08527-5

Figure Lengend Snippet: Phospho-SMAD2/3 levels detected by Western blotting in ( a ) control group and ( b ) IGFBP6-knockout group treated with TGF-β (10 ng/mL) for 15 min, 30 min, or 60 min with or without PA (10 µM) ( n = 3). Data are expressed as means ± SD, One-way ANOVA. c Immunofluorescence analysis of TGF-β (10 ng/mL) -induced SMAD2/3 nuclear translocation in LX2 cells with or without IGFBP6 knockout, treated with or without PA (10 µM) (scale bar: 25 μm). d The mRNA expression levels of Acta2 , Fn1 , and Col1a1 in primary mouse HSCs in the presence or absence of PA or the SMAD3 inhibitor SIS3 were detected by qRT-PCR ( n = 3). Data are expressed as means ± SD, Two-way ANOVA.

Article Snippet: Recombinant human IGFBP6 protein with His-tag (MCE, HY- P73141 ) was purified using Ni-NTA affinity chromatography (Thermo Scientific, HisPur).

Techniques: Western Blot, Control, Knock-Out, Immunofluorescence, Translocation Assay, Expressing, Quantitative RT-PCR