Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Release of IGFBP5 in SASP and its effect on senescence. A Representative micrographs of MSCs stained to identify nuclei (DAPI), Ki67 (red), and to evaluate β-galactosidase activity (dark gray). The white arrows indicate senescent cells, which are β-galactosidase positive (β-gal +) and Ki67 negative (Ki67-). We employed a Leica CTR500 microscope, which was equipped with a DCF3000G digital monochrome camera. The β-galactosidase activity was captured as a gray-stain using this configuration. This experimental method allowed us to identify cells that exhibited a visible light signal β-galactosidase along with others expressing fluorescent signals within the same cell. CT: untreated cells; CM-IR: cells treated with conditioned medium (CM) collected from irradiated (IR) cells; CM-IR + ab αIGFBP5: cells treated with CM in the presence of IGFBP5 neutralizing antibodies; rIGFBP5: cells incubated with recombinant IGFBP5. The scale bar corresponds to 100 microns. The graph shows the percentage of senescent cells under different experimental conditions. The symbols *** p < 0.001 and * p < 0.05 indicate statistical significance between the control (CT) and treated samples. The symbol ## p < 0.01 indicates statistical significance between the CM-IR sample, chosen as a reference, and CM-IR + ab αIGFBP5. B The graph shows the level of IGFBP5 in CM of MSCs 24 h following irradiation (IR). IR: irradiated MSCs; Anti-oxi: the irradiated MSCs were treated with an anti-oxidant mixture; PXB: irradiated cells were incubated with a drug inhibiting COX2 activity. The symbol ** p < 0.01 indicates statistical significance between the control (CT) and irradiated samples. The symbols ## p < 0.01 and # p < 0.05 indicate statistical significance between the IR sample (second column), chosen as a reference, and IR + Anti-oxi or IR + PXB. The western blot under the graph shows a representative image of IGFBP5 immunodetection. C The graph shows the percentage of senescent cells (β-galactosidase positive and Ki67 negative) under different experimental conditions. rIGFBP5: cells incubated with recombinant IGFBP5; IGFII: cell treated with IGFII either in presence or absence of antibody against IGFIIR (abα IGFIIR); siLRP1: siRNA targeting LRP1 mRNA; siCAV1: siRNA targeting CAVEOLIN-1; siCTR: control siRNA. The symbol ** p < 0.01 indicates statistical significance between the control (first column) and samples (from second to fourth columns) treated with rIGFBP5 and/or IGFII. The symbol °°° p < 0.001 indicates statistical significance between cells treated with abα IGFIIR alone (chosen as reference, see fifth column) and the other samples treated with the antibody in presence of rIGFBP5 and/or IGFII (from sixth to eight columns). The symbols §§§ p < 0.001 indicate and § p < 0.05 indicate statistical significance between the sample treated with siCT (chosen as the reference, see ninth column) and the other siRNA treated samples, reported in columns from the 10th to 12th. The symbol ## p < 0.01 indicates the statistical difference between the sample treated with genistein (Gen) in presence of IGFBP5 and the one treated with IGFBP5 only (second column from left), chosen as reference

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Staining, Activity Assay, Microscopy, Expressing, Irradiation, Incubation, Recombinant, Control, Western Blot, Immunodetection

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: IGFBP5 internalization. A Representative images of MSCs incubated with His-tag-IGFBP5 (IGFBP5-HIS) and stained to identify nuclei (DAPI) and His-tag-IGFBP5 (red). Additionally, CAVEOLIN-1 (CAV1), LRP1, or ITGA2 were stained green. The pictures were taken at 1, 3, and 5 min after IGFBP5 incubation. The inset shows magnified images of IGFBP5 and CAVEOLIN-1 co-localization. The scale bar corresponds to 100 microns. B Representative images of MSCs incubated with His-tag-IGFBP5 and stained to identify nuclei (DAPI) and His-tag-IGFBP5 (green). The pictures were taken at 0, 0.5, 1, 3, 10, 30 and 60 min after IGFBP5 incubation. The scale bar corresponds to 100 microns. C Representative images of Duolink assay to identify physical proximity between IGFBP5 and CAVEOLIN-1. The red staining indicates a close interaction between IGFBP5 and CAVEOLIN-1 5 min following MSCs incubation with His-tag-IGFBP5. The nuclei were stained with DAPI (blue). The scale bar corresponds to 100 microns. D Representative images of MSCs incubated with His-tag-IGFBP5, either in presence or absence of Brefeldin A, and stained to identify nuclei (DAPI) and His-tag-IGFBP5 (red). Additionally, GOLGB1 was stained green. The pictures were taken at 3 min after IGFBP5 incubation. The scale bar corresponds to 100 microns

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Incubation, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Western blot analysis of IGFBP5. Experiments were carried out by cell fractionation into cytoplasmic (cyto) and nuclear fractions. The image shows IGFBP5 levels at different time points following the incubation of MSCs culture with His-tagged IGFBP5. Three different primary antibodies against IGFBP5 were used (see main text). GAPDH and Histone H4 were used as cytoplasmic and nuclear markers, respectively

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Western Blot, Cell Fractionation, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Paracrine action of IGFBP5. A MSCs transfected with control siRNA or IGFBP5-siRNA were X-ray irradiated, and senescence was evaluated 48 h later. The graph depicts the percentage of senescent cells under different experimental conditions. The symbols *** p < 0.001 and * p < 0.05 indicate statistical significance between the control (siCT) and other samples. The # ( p < 0.05) indicateds statistical significance between irradiated siCT versus irradiated siIGFBP5 ( B ) Healthy MSCs were incubated for 48 h with conditioned media (CM) obtained from the previously mentioned samples. The graph illustrates the percentage of senescent cells under different experimental conditions. The symbol *** p < 0.001 indicates statistical significance between the control (siCT) and other samples. The ## ( p < 0.01) indicateds statistical significance between irradiated siCT versus irradiated siIGFBP5. C Representative images of cells stained with anti-γH2AX (green) and His-tag IGFBP5 (red) are shown. Cell nuclei were stained with DAPI. The β-galactosidase activity was evidenced as dark gray. We employed a Leica CTR500 microscope, which was equipped with a DCF3000G digital monochrome camera. The β-galactosidase activity was captured as a gray-stain using this configuration. The arrows show cells that were β-galactosidase/γH2AX positive and IGFBP5 negative

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Transfection, Control, Irradiation, Incubation, Staining, Activity Assay, Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Interaction between IGFBP5 and retinoic acid receptors. A Cell lysates were immunoprecipitated with either anti-RXRα or anti-RARα antibodies and then subjected to western blot analysis using anti-IGFBP5 antibody. Reciprocal immunoprecipitation (IP) was performed with anti-IGFBP5 antibody, followed by western blots (WB) using either anti-RXRα or anti-RARα antibodies. P and Sup refer to the pellet and supernatant, respectively, of the immunoprecipitation reaction. B Representative images of the Duolink assay to identify the physical proximity between IGFBP5 and RARα. The red staining indicates a close interaction between IGFBP5 and RARα ten minutes after MSCs were incubated with His-tagged IGFBP5. The nuclei were stained with DAPI (blue). The scale bar corresponds to 100 microns. C Recombinant RARα, immobilized on protein A beads, was incubated with IGFBP5 in the presence or absence of ATRA, followed by western blot analysis using anti-IGFBP5 antibody. P and Sup refer to the pellet and supernatant, respectively, of the reaction. D The graph shows the percentage of senescent cells following incubation with IGFBP5 under different experimental conditions. The symbol *** p < 0.001 indicates statistical significance between untreated cells and samples incubated with IGFBP5 (first and second column). The symbols ### p < 0.001 and ## p < 0.01 indicate the statistically significance between the sample treated with IGFBP5 (second column from left), which was chosen as reference, and the others with IGFBP5 in combination with further treatments. E Fluorescence quenching assay. The graphs show the ultraviolet peak emission of IGFBP5 and RARα, due to tryptophan, phenylalanine, and tyrosine, either in the absence or presence of increasing amounts of ATRA, which acted as the quencher. Data are reported with standard deviation. The symbols * p < 0.05, ** p < 0.01 indicate statistical significance between samples incubated with and without ATRA. The latter condition was chosen as the reference

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Immunoprecipitation, Western Blot, Staining, Incubation, Recombinant, Fluorescence, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: Serum IGFBP5 in patients undergoing medical irradiation. The picture illustrates the ELISA analysis of IGFBP5 in the sera of 10 patients before and 48 h after an abdominal CT scan. Each patient is identified by a number. The data are expressed as arbitrary units (A.U.). For every patient, the significant difference between samples harvested before and after CT is denoted with ** ( p < 0.01) or * ( p < 0.05)

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Computed Tomography

Journal: Cell Communication and Signaling : CCS

Article Title: IGFBP5 is released by senescent cells and is internalized by healthy cells, promoting their senescence through interaction with retinoic receptors

doi: 10.1186/s12964-024-01469-1

Figure Lengend Snippet: IGFBP5 signaling in senescence. Healthy cells may become senescent cells following genotoxic stress and then release SASP, which contains IGFBP5. The paracrine action of IGFBP5 may induce secondary senescence in healthy cells not directly affected by genotoxic injury. IGFBP5 can enter cell nuclei through caveolae-dependent endocytosis. Within nuclei, IGFBP5 can interact with RAR/RXR heterodimers and contribute to the transcriptional regulation of genes involved in the executive senescence program

Article Snippet: To assess the pathways involved in IGFBP5 intake, MSCs were incubated with rIGFBP5-His Tag (PKSH032598, Elabscience TX, USA) at different time points while inhibiting different endocytosis-related pathways.

Techniques:

Journal: Andrology

Article Title: IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice

doi: 10.1111/andr.13234

Figure Lengend Snippet: Increased IGFBP5 expression under HG or diabetic conditions. (A) IGFBP5 gene expression was evaluated in MCECs exposed to NG or HG conditions. (B–D) Representative Western blots of IGFBP5 in MCECs and MCEC culture media after exposure to NG or HG conditions in vitro and in age‐matched control and diabetic mouse penile tissue in vivo. (E and F) IGFBP5 (red) or PECAM1 (green, an endothelial cell marker) immunostaining in MCECs and mouse cavernosum tissue. Nuclei were labeled with DAPI. The scale bars indicate 50 μm for MCECs and 100 μm for cavernosum tissue. (G–J) Normalized relative band intensity values as determined using ImageJ ( n = 4, ** p < 0.01, *** p < 0.001). (K) IGFBP5‐immunopositive areas in MCECs under NG or HG condition were quantified using ImageJ ( n = 4, *** p < 0.001). (L) IGFBP5‐immunopositive areas in PECAM1 expressing cells were quantified using ImageJ ( n = 4, *** p < 0.001). Data in graphs are presented as means ± SEMs. The value expressed as ratios of the NG or Control group was set to 1. DAPI, 4,6‐diamidino‐2‐phenylindole; DM, STZ‐induced diabetes; HG, high glucose; MCECs, mouse cavernous endothelial cells; NG, normal glucose

Article Snippet: In order to examine the effect of recombinant mouse IGFBP5 (rmIGFBP5), MCECs were treated with rmIGFBP5 (500 ng/ml; Mybiosource Inc., San Diego, CA, USA) under HG conditions for 72 h. To characterize cell types, the cells were stained with antibodies to hamster monoclonal anti‐PECAM1 antibody (an endothelial cell marker, 1:50, Millipore, Temecula, CA, USA), rabbit polyclonal anti‐NG2 antibody (a pericyte marker, 1:50, Millipore), mouse monoclonal anti‐CD90 (a fibroblast marker, 1:50, R&D Systems Inc.), rabbit polyclonal anti‐smooth muscle α‐actin (α‐SMA, a smooth muscle cell marker, 1:100, Sigma‐Aldrich), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Expressing, Gene Expression, Western Blot, In Vitro, Control, In Vivo, Marker, Immunostaining, Labeling

Journal: Andrology

Article Title: IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice

doi: 10.1111/andr.13234

Figure Lengend Snippet: Knockdown of IGFBP5 restored the angiogenic activity of endothelial cells in vitro and ex vivo. (A and B) Representative data for IGFBP5 knockdown by IGFBP5 siRNA transfection or IGFBP5 shRNA lentivirus infection (5 × 10 3 , 1 × 10 4 , 2.5 × 10 4 , or 5 × 10 4 IFU/ml of culture medium) in mouse cavernous endothelial cells (MCECs). (C and D) Tube formation and migration assays in primary cultured MCECs exposed to NG or HG conditions and treated with rmIGFBP5 protein (0.5 μg/ml), Sc, or siIGFBP5. (E) BrdU incorporation assay of primary cultured MCECs exposed to NG or HG conditions treated with Sc or siIGFBP5. Nuclei were labeled with DAPI (blue). Scale bars indicate 100 μm. (F) Aortic ring assay in mouse aortas exposed to NG or HG conditions and treated with lentiviral particles of shCon or shIGFBP5. (G and I) Numbers of master junctions per field ( n = 4, ** p < 0.01, *** p < 0.001). 40X magnification. (H and J) Migrated cells were quantified using ImageJ ( n = 4, *** p < 0.001). (K) Numbers of BrdU‐immunopositive endothelial cells were quantified using ImageJ ( n = 4, *** p < 0.001). (L) Areas of microvessels growing from aortic rings were also quantified using ImageJ ( n = 4, * p < 0.05, *** p < 0.001). Data in graphs are presented as means ± SEMs. The value expressed as ratios of the NG: PBS, Sc or shCon group was set to 1. BrdU, bromodeoxyuridine; DAPI, 4.6‐diamidino‐2‐phenylindole; IFU, infection units; ns, not significant; Sc, scrambled siRNA; shCon, scrambled shRNA; shRNA, short hairpin RNA; shIGFBP5, shRNA for IGFBP5; siIGFBP5, siRNA for IGFBP5; siRNA, small interfering RNA

Article Snippet: In order to examine the effect of recombinant mouse IGFBP5 (rmIGFBP5), MCECs were treated with rmIGFBP5 (500 ng/ml; Mybiosource Inc., San Diego, CA, USA) under HG conditions for 72 h. To characterize cell types, the cells were stained with antibodies to hamster monoclonal anti‐PECAM1 antibody (an endothelial cell marker, 1:50, Millipore, Temecula, CA, USA), rabbit polyclonal anti‐NG2 antibody (a pericyte marker, 1:50, Millipore), mouse monoclonal anti‐CD90 (a fibroblast marker, 1:50, R&D Systems Inc.), rabbit polyclonal anti‐smooth muscle α‐actin (α‐SMA, a smooth muscle cell marker, 1:100, Sigma‐Aldrich), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Knockdown, Activity Assay, In Vitro, Ex Vivo, Transfection, shRNA, Infection, Migration, Cell Culture, BrdU Incorporation Assay, Labeling, Aortic Ring Assay, Small Interfering RNA

Journal: Andrology

Article Title: IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice

doi: 10.1111/andr.13234

Figure Lengend Snippet: Knockdown of IGFBP5 improved erectile dysfunction in mice with STZ‐induced diabetes (DM). (A) Representative ICP responses for age‐matched nondiabetic controls, diabetic mice stimulated at 2 weeks after an intracavernous PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5×10 4 IFU/mouse for shRNA lentiviral particles) injection. The cavernous nerve was stimulated at 5 V, and stimulus time is indicated by a solid bar. (B and C) Ratios of mean maximal ICP and total ICP (area under the curve) versus MSBP were calculated for each group ( n = 6, *** p < 0.001). Data in graphs are presented as means ± SEMs. DM, STZ‐induced diabetes; ICP, intracavernous pressure; MSBP, mean systolic blood pressure; ns, not significant; shCon, scrambled shRNA; shIGFBP5, shRNA for IGFBP5; shRNA, short hairpin RNA; STZ, streptozotocin;

Article Snippet: In order to examine the effect of recombinant mouse IGFBP5 (rmIGFBP5), MCECs were treated with rmIGFBP5 (500 ng/ml; Mybiosource Inc., San Diego, CA, USA) under HG conditions for 72 h. To characterize cell types, the cells were stained with antibodies to hamster monoclonal anti‐PECAM1 antibody (an endothelial cell marker, 1:50, Millipore, Temecula, CA, USA), rabbit polyclonal anti‐NG2 antibody (a pericyte marker, 1:50, Millipore), mouse monoclonal anti‐CD90 (a fibroblast marker, 1:50, R&D Systems Inc.), rabbit polyclonal anti‐smooth muscle α‐actin (α‐SMA, a smooth muscle cell marker, 1:100, Sigma‐Aldrich), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Knockdown, shRNA, Injection

Journal: Andrology

Article Title: IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice

doi: 10.1111/andr.13234

Figure Lengend Snippet: IGFBP5 knockdown increased cavernous endothelial cell, pericyte, and nNOS‐expressing neuronal cell contents in mice with STZ‐induced diabetes (DM). (A) PECAM1 (an endothelial cell marker, green) or NG2 (a pericyte marker, red) immunostaining in cavernous tissues from age‐matched nondiabetic controls and diabetic mice stimulated 2 weeks after intracavernous a PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5×10 4 IFU/mice for shRNA lentiviral particles) injection. Scale bar indicates 100 μm. (B) nNOS (a neuronal cell marker, red) immunostaining in the dorsal nerve bundles of an age‐matched nondiabetic control and a diabetic mouse stimulated at 2 weeks after receiving intracavernous PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5×10 4 IFU/mice for shRNA lentiviral particles) injections. Nuclei were labeled with DAPI (blue). Scale bar indicates 25 μm. (C–E) Quantifications of cavernous endothelial cells, pericytes, or nNOS‐expressing neuronal cells by ImageJ ( n = 6, *** p < 0.001). Data in graphs are presented as means ± SEMs. The value expressed as ratios of the Control group was set to 1. DAPI = 4.6‐diamidino‐2‐phenylindole; DM, STZ‐induced diabetes; ns, not significant; shCon, scrambled shRNA; shIGFBP5, shRNA for IGFBP5; shRNA, short hairpin RNA

Article Snippet: In order to examine the effect of recombinant mouse IGFBP5 (rmIGFBP5), MCECs were treated with rmIGFBP5 (500 ng/ml; Mybiosource Inc., San Diego, CA, USA) under HG conditions for 72 h. To characterize cell types, the cells were stained with antibodies to hamster monoclonal anti‐PECAM1 antibody (an endothelial cell marker, 1:50, Millipore, Temecula, CA, USA), rabbit polyclonal anti‐NG2 antibody (a pericyte marker, 1:50, Millipore), mouse monoclonal anti‐CD90 (a fibroblast marker, 1:50, R&D Systems Inc.), rabbit polyclonal anti‐smooth muscle α‐actin (α‐SMA, a smooth muscle cell marker, 1:100, Sigma‐Aldrich), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Knockdown, Expressing, Marker, Immunostaining, shRNA, Injection, Control, Labeling

Journal: Andrology

Article Title: IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice

doi: 10.1111/andr.13234

Figure Lengend Snippet: Knockdown of IGFBP5 induced cavernous eNOS Ser 1177 phosphorylation and reduced cavernous endothelial cell apoptosis in mice with STZ‐induced diabetes (DM). (A) PECAM1 (green) and p‐eNOS Ser 1177 (red) immunostaining in cavernous tissues from age‐matched nondiabetic control and diabetic mice stimulated at 2 weeks after receiving an intracavernous PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5 × 10 4 IFU/mice for shRNA lentiviral particles) injection. The scale bar indicates 100 μm. (B) TUNEL (green) and PECAM1 (red) immunostaining in cavernous tissues from age‐matched nondiabetic controls and diabetic mice stimulated at 2 weeks after receiving an intracavernous PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5 × 10 4 IFU/mice for shRNA lentiviral particles) injection. Nuclei were labeled with DAPI (blue). The scale bar indicates 100 μm. (C) Representative Western blots of p‐eNOS Ser 1177 and eNOS in the control and three experimental groups. (D) Quantitative analysis of p‐eNOS Ser 1177 immunopositive areas by ImageJ ( n = 5, *** p < 0.001) (E) Normalized band intensity values for p‐eNOS Ser 1177 and eNOS were quantified using ImageJ ( n = 5, *** p < 0.001). The value expressed as ratios of the Control group was set to 1. (F) Number of apoptotic cells were quantified by ImageJ ( n = 6, *** p < 0.001). Data in graphs are presented as means ± SEMs. DAPI = 4.6‐diamidino‐2‐phenylindole; DM, STZ‐induced diabetes; eNOS, endothelial nitric oxide synthase; NOS, nitric oxide synthase; p‐eNOS, phospho‐endothelial nitric oxide synthase; shCon, scrambled shRNA; shIGFBP5, shRNA for IGFBP5; shRNA, short hairpin RNA; ns, not significant; TUNEL, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling

Article Snippet: In order to examine the effect of recombinant mouse IGFBP5 (rmIGFBP5), MCECs were treated with rmIGFBP5 (500 ng/ml; Mybiosource Inc., San Diego, CA, USA) under HG conditions for 72 h. To characterize cell types, the cells were stained with antibodies to hamster monoclonal anti‐PECAM1 antibody (an endothelial cell marker, 1:50, Millipore, Temecula, CA, USA), rabbit polyclonal anti‐NG2 antibody (a pericyte marker, 1:50, Millipore), mouse monoclonal anti‐CD90 (a fibroblast marker, 1:50, R&D Systems Inc.), rabbit polyclonal anti‐smooth muscle α‐actin (α‐SMA, a smooth muscle cell marker, 1:100, Sigma‐Aldrich), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Knockdown, Phospho-proteomics, Immunostaining, Control, shRNA, Injection, TUNEL Assay, Labeling, Western Blot, End Labeling

Journal: Andrology

Article Title: IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice

doi: 10.1111/andr.13234

Figure Lengend Snippet: Knockdown of IGFBP5 restored cell‐to‐cell junction protein levels and reduced oxidized LDL extravasation in diabetic mice. (A) Occludin (green) and claudin‐5 (red) immunostaining in cavernous tissues of age‐matched nondiabetic controls and diabetic mice stimulated at 2 weeks after receiving an intracavernous PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5 × 10 4 IFU/mice for shRNA lentiviral particles) injection. The scale bar indicates 100 μm. (B) Ox‐LDL (red) and PECAM1 (green) immunostaining in cavernous tissues from age‐matched non‐diabetic controls and diabetic mice stimulated 2 weeks after receiving an intracavernous PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5 × 10 4 IFU/mice for shRNA lentiviral particles) injection. The scale bar indicates 100 μm. Nuclei were labeled with DAPI (blue). (C and D) Quantitative analysis of occludin and claudin‐5 immunopositive areas as determined by ImageJ ( n = 5, *** p < 0.001). (E) Quantitative analysis of Ox‐LDL immunopositive areas by ImageJ ( n = 5, *** p < 0.001). Ratios are expressed versus non‐diabetic controls. Data in graphs are presented as means ± SEMs. DAPI = 4.6‐diamidino‐2‐phenylindole; DM, STZ‐induced diabetes; ns, not significant; Ox‐LDL, oxidized low‐density lipoprotein; shCon, scrambled shRNA; shIGFBP5, shRNA for IGFBP5; shRNA, short hairpin RNA

Article Snippet: In order to examine the effect of recombinant mouse IGFBP5 (rmIGFBP5), MCECs were treated with rmIGFBP5 (500 ng/ml; Mybiosource Inc., San Diego, CA, USA) under HG conditions for 72 h. To characterize cell types, the cells were stained with antibodies to hamster monoclonal anti‐PECAM1 antibody (an endothelial cell marker, 1:50, Millipore, Temecula, CA, USA), rabbit polyclonal anti‐NG2 antibody (a pericyte marker, 1:50, Millipore), mouse monoclonal anti‐CD90 (a fibroblast marker, 1:50, R&D Systems Inc.), rabbit polyclonal anti‐smooth muscle α‐actin (α‐SMA, a smooth muscle cell marker, 1:100, Sigma‐Aldrich), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Knockdown, Immunostaining, shRNA, Injection, Labeling

Journal: Andrology

Article Title: IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice

doi: 10.1111/andr.13234

Figure Lengend Snippet: Knockdown of IGFBP5 enhanced AKT and ERK signaling and inhibited p38 signaling in diabetic mice. (A) Representative western blots for phospho‐AKT/AKT, phospho‐ERK/ERK, phospho‐p38/p38, and IGFBP5 in cavernous tissues from nondiabetic controls and diabetic mice stimulated 2 weeks after receiving an intracavernous PBS, shCon‐, or shIGFBP5‐lentivirus (20 μl for PBS, 5 × 10 4 IFU/mice for shRNA lentiviral particles) injection. (B and E) Data are presented as relative intensities of p‐AKT, p‐ERK, p‐p38, and IGFBP5 proteins versus AKT, ERK, p38, and β‐actin, respectively, as determined by ImageJ ( n = 4, *** p < 0.001). The value expressed as ratios of the Control group was set to 1. Data in graphs are presented as means ± SEMs. DM, STZ‐induced diabetes; ns, not significant; shCon, scrambled shRNA; shIGFBP5, shRNA for IGFBP5; shRNA, short hairpin RNA

Article Snippet: In order to examine the effect of recombinant mouse IGFBP5 (rmIGFBP5), MCECs were treated with rmIGFBP5 (500 ng/ml; Mybiosource Inc., San Diego, CA, USA) under HG conditions for 72 h. To characterize cell types, the cells were stained with antibodies to hamster monoclonal anti‐PECAM1 antibody (an endothelial cell marker, 1:50, Millipore, Temecula, CA, USA), rabbit polyclonal anti‐NG2 antibody (a pericyte marker, 1:50, Millipore), mouse monoclonal anti‐CD90 (a fibroblast marker, 1:50, R&D Systems Inc.), rabbit polyclonal anti‐smooth muscle α‐actin (α‐SMA, a smooth muscle cell marker, 1:100, Sigma‐Aldrich), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Knockdown, Western Blot, shRNA, Injection, Control