Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A , B Tumor spheroid invasion in collagen matrix after ( A ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( B ) treatment with MEL23. Representative images above and quantification below of the area invaded and the number of cells invading 24 h after implantation. C , D Tumor spheroid invasion in the collagen-BME matrix after ( C ) transfection of HT1080 p53KO cells with siRNAs against Mdm2 or ( D ) treatment with MEL23. Representative images and quantification of the area invaded 24 h after implantation. Graphs show three pooled independent experimental replicates, the total number of spheroids in each condition is: siCtrl = 10, siMdm2#1 = 15, siMdm2#2 = 12 in panel ( A ); DMSO = 25, MEL23 = 24 in panel ( B ); siCtrl = 12, siMdm2#1 = 14, siMdm2#2 = 15 in panel ( C ), and DMSO = 8, MEL23 = 9 in panel ( D ). E Representative images of cell morphology of HT1080 p53KO cells transfected with siRNAs against Mdm2 or siCtrl in collagen matrix, 6 h after implantation. F Quantification of more circular cells (circularity of 0.75 or higher) in each condition shown in ( E ), n = 3 groups. The graph represents the average fold change of more circular morphology in Mdm2 silenced cells compared to control in three independent experimental replicates. More details about the quantification can be found in the method’s session. G – I HT1080 p53KO cells stably expressing shRNA scramble (shScramble) or a pool of Mdm2 shRNAs (shMdm2) were used to analyze metastatic burden using mouse models. G Protein levels of Mdm2 and MdmX in HT1080 shScramble and shMdm2 stable cell lines. β-actin was used as a loading control. H , I Representative images above and quantification below of metastatic foci in the lungs after implantation of shScramble or shMdm2 cells using ( H ) orthotropic model, n = 4 mice/group or ( I ) tail-vein model, n = 8 mice/group. In all box and whisker plots the boxes extend from 25 to 75 percentiles and whiskers show min and max values, The line in the center of each box represents the median. Graphs shown in panels ( F , H , I ) represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: For shSpry4 stable cell lines, cells were transduced with a set of four shRNAs against Spry4 (OriGene, cat.#HC108594) or shRNA negative control (OriGene, cat.#TR30033), and using the appropriate drug for cell selection.

Techniques: Transfection, Control, Stable Transfection, Expressing, shRNA, Whisker Assay

Journal: Nature Communications

Article Title: Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

doi: 10.1038/s41467-024-51488-2

Figure Lengend Snippet: A – D HT1080 p53KO cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. A Protein levels of Mdm2, MdmX, and Spry4 after transfection with indicated siRNAs, n = 3 samples. β-actin was used as a loading control. B Quantification of wound scratch migration assay in cells treated with the indicated siRNAs as in Fig. , n = 3 samples. C Quantification of cell area after attachment to collagen-coated coverslips as in Fig. , n = 3 samples. D Immunofluorescence showing FA foci by vinculin staining (red), cell surface was outlined by phalloidin staining (green), and nuclei (blue) as detected by DAPI staining, n = 3 groups. Representative images are shown on the left, and the quantification of FA parameters is shown on the right. In ( C , D ) the graphs shown represent mean ± SD of independent experimental replicates and in each replicate all parameters were quantified in at least 20 events/condition for total of at least 60 events/condition. E , F H1299 cells were transfected with siRNA against Mdm2 alone or both siRNA against Mdm2 and a pool of Spry4 siRNAs. E Protein levels of Mdm2, MdmX, and Spry4 after transfection with indicated siRNAs. β-actin was used as a loading control, n = 3 samples. F Quantification of wound scratch migration assay comparing migration into wound scratches in cells treated with the indicated siRNAs as in Fig. , n = 3 samples. G , H HT1080 p53KO cell lines were established stably expressing a pool of shRNAs against Mdm2 alone or Mdm2 and Spry4 together. G Protein levels of Mdm2, MdmX, and Spry4 in stable cell lines. β-actin was used as a loading control, n = 3 samples. H Analysis of metastatic burden in vivo using tail-vein injection model as in Fig. H, . Representative images above and quantification below of metastatic foci in the lungs after 8 weeks of injection, n = 7 mice/group. The graphs shown represent the mean ± SD of independent experimental replicates. More details about the statistical tests used can be found in the Source Data file.

Article Snippet: For shSpry4 stable cell lines, cells were transduced with a set of four shRNAs against Spry4 (OriGene, cat.#HC108594) or shRNA negative control (OriGene, cat.#TR30033), and using the appropriate drug for cell selection.

Techniques: Transfection, Control, Migration, Immunofluorescence, Staining, Stable Transfection, Expressing, In Vivo, Injection

Journal: Molecular Medicine Reports

Article Title: Lidocaine inhibits the progression of retinoblastoma in vitro and in vivo by modulating the miR-520a-3p/EGFR axis

doi: 10.3892/mmr.2019.10363

Figure Lengend Snippet: Lidocaine upregulates the expression of miR-520a-3p in RB cells. (A) Expression of miR-520a-3p in RB and normal adjacent tissues. n=30/group. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal tissues. (B) Expression of miR-520a-3p in RB cell lines and the retinal pigment epithelial cell line ARPE-19. *P<0.05, **P<0.01 vs. ARPE-19. (C) Expression of miR-520a-3p in Y79 cells following treatment with 500 µM lidocaine for 24 h. **P<0.01 vs. control. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. miR-520a-3p, microRNA-520a-3p; RB, retinoblastoma.

Article Snippet: When the cell confluence reached 60–70%, the Y79 cells were transfected with 100 nM miR-520a-3p inhibitor (inhibitor) (cat. no. miR20002834-1-5; Guangzhou RiboBio Co., Ltd., Guangzhou, China), 100 nM miR-520a-3p mimic (mimic) (cat. no. miR10002834-1-5; Guangzhou RiboBio Co., Ltd.) or 100 nM miR-520a-3p scramble [negative control (NC; cat. no. miRB160401025525-2-1; Guangzhou RiboBio Co., Ltd.) using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.)] for 24 h according to the manufacturer's protocols.

Techniques: Expressing, Standard Deviation, Control

Journal: Molecular Medicine Reports

Article Title: Lidocaine inhibits the progression of retinoblastoma in vitro and in vivo by modulating the miR-520a-3p/EGFR axis

doi: 10.3892/mmr.2019.10363

Figure Lengend Snippet: EGFR is a direct target gene of miR-520a-3p. (A) TargetScan prediction of a binding site for miR-520a-3p in the EGFR 3′-UTR. (B) Luciferase activity in Y79 following co-transfection with mimic or NC and luciferase reporter plasmids containing WT-EGFR or MUT-EGFR. **P<0.01 vs. NC. Relative expression of (C) miR-520a-3p, and (D) EGFR mRNA and (E) protein in Y79 cells following transfection with NC or mimics. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control. EGFR, epidermal growth factor receptor; 3′-UTR, 3′-untranslated region; miR-520a-3p, microRNA-520a-30; MUT-EGFR, mutated EGFR 3′-UTR; WT-EGFR, wild-type EGFR 3′-UTR; NC, negative control; mimic, miR-520a-3p mimic.

Article Snippet: When the cell confluence reached 60–70%, the Y79 cells were transfected with 100 nM miR-520a-3p inhibitor (inhibitor) (cat. no. miR20002834-1-5; Guangzhou RiboBio Co., Ltd., Guangzhou, China), 100 nM miR-520a-3p mimic (mimic) (cat. no. miR10002834-1-5; Guangzhou RiboBio Co., Ltd.) or 100 nM miR-520a-3p scramble [negative control (NC; cat. no. miRB160401025525-2-1; Guangzhou RiboBio Co., Ltd.) using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.)] for 24 h according to the manufacturer's protocols.

Techniques: Binding Assay, Luciferase, Activity Assay, Cotransfection, Expressing, Transfection, Standard Deviation, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: Lidocaine inhibits the progression of retinoblastoma in vitro and in vivo by modulating the miR-520a-3p/EGFR axis

doi: 10.3892/mmr.2019.10363

Figure Lengend Snippet: Effects of lidocaine on the expression of miR-520a-3p and EGFR in retinoblastoma cells. Y79 cells were transfected with NC or inhibitor for 24 h, and then treated with lidocaine or control for 24 h. Expression of (A) miR-520a-3p, and (B) EGFR mRNA and (C) protein in the various groups. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control; && P<0.01 vs. 500 µM lidocaine. miR-520a-3p, microRNA-520a-3p; NC, negative control; inhibitor, miR-520a-3p inhibitor; EGFR, epidermal growth factor receptor.

Article Snippet: When the cell confluence reached 60–70%, the Y79 cells were transfected with 100 nM miR-520a-3p inhibitor (inhibitor) (cat. no. miR20002834-1-5; Guangzhou RiboBio Co., Ltd., Guangzhou, China), 100 nM miR-520a-3p mimic (mimic) (cat. no. miR10002834-1-5; Guangzhou RiboBio Co., Ltd.) or 100 nM miR-520a-3p scramble [negative control (NC; cat. no. miRB160401025525-2-1; Guangzhou RiboBio Co., Ltd.) using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.)] for 24 h according to the manufacturer's protocols.

Techniques: Expressing, Transfection, Control, Standard Deviation, Negative Control

Journal: Molecular Medicine Reports

Article Title: Lidocaine inhibits the progression of retinoblastoma in vitro and in vivo by modulating the miR-520a-3p/EGFR axis

doi: 10.3892/mmr.2019.10363

Figure Lengend Snippet: Lidocaine inhibits the proliferation and promotes the apoptosis of retinoblastoma cells via upregulation of miR-520a-3p. Y79 cells were transfected with NC or inhibitor for 24 h, and then treated with lidocaine or control for 24 h. (A) Proliferation of Y79 cells as determined by a Cell Counting Kit-8 assay. (B) Apoptosis of Y79 cells as determined by a flow cytometry assay. (C) Early apoptosis Early apoptosis (Q4) and late apoptosis (Q2) were calculated and the apoptosis rate was presented. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control; && P<0.01 vs. 500 µM lidocaine. NC, negative control; miR-520a-3p, microRNA-520a-3p; inhibitor, miR-520a-3p inhibitor; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: When the cell confluence reached 60–70%, the Y79 cells were transfected with 100 nM miR-520a-3p inhibitor (inhibitor) (cat. no. miR20002834-1-5; Guangzhou RiboBio Co., Ltd., Guangzhou, China), 100 nM miR-520a-3p mimic (mimic) (cat. no. miR10002834-1-5; Guangzhou RiboBio Co., Ltd.) or 100 nM miR-520a-3p scramble [negative control (NC; cat. no. miRB160401025525-2-1; Guangzhou RiboBio Co., Ltd.) using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.)] for 24 h according to the manufacturer's protocols.

Techniques: Transfection, Control, Cell Counting, Flow Cytometry, Standard Deviation, Negative Control

Journal: Molecular Medicine Reports

Article Title: Lidocaine inhibits the progression of retinoblastoma in vitro and in vivo by modulating the miR-520a-3p/EGFR axis

doi: 10.3892/mmr.2019.10363

Figure Lengend Snippet: Lidocaine reduces the volume and weight of RB tumors in vivo via upregulation of miR-520a-3p. First, the level of miR-520a-3p and EGFR in RB model mice and normal control mice was detected, and RB tissues from the RB model mice (Cancer tissues) and the normal control mice (Normal tissues) were collected. Expression of (A) miR-520a-3p, and (B) EGFR mRNA and (C) protein in RB and adjacent tissues of mice. **P<0.01 vs. normal tissues. Tumor xenografts were implanted in mice via subcutaneous injection of 2×10 6 Y79 cells suspended in 100 µl serum-free RPMI-1640 medium on the sides of the posterior flank of nude mice. At 18 days after tumor inoculation, mice were subsequently treated with lidocaine (1.5 mg/kg) via tail vein injection. Y79 cells were transfected with NC or inhibitor prior to subcutaneous injection. Expression of (D) miR-520a-3p, and (E) EGFR mRNA and (F) protein in tumor tissues 18 days after tumor inoculation. (G) Tumor volume and (H) weight in the various groups. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. *P<0.05, **P<0.01 vs. control; & P<0.05, && P<0.01 vs. lidocaine. RB, retinoblastoma; miR-520a-3p, microRNA-520a-3p; EGFR, epidermal growth factor receptor; inhibitor, miR-520a-3p inhibitor; NC, negative control.

Article Snippet: When the cell confluence reached 60–70%, the Y79 cells were transfected with 100 nM miR-520a-3p inhibitor (inhibitor) (cat. no. miR20002834-1-5; Guangzhou RiboBio Co., Ltd., Guangzhou, China), 100 nM miR-520a-3p mimic (mimic) (cat. no. miR10002834-1-5; Guangzhou RiboBio Co., Ltd.) or 100 nM miR-520a-3p scramble [negative control (NC; cat. no. miRB160401025525-2-1; Guangzhou RiboBio Co., Ltd.) using Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.)] for 24 h according to the manufacturer's protocols.

Techniques: In Vivo, Control, Expressing, Injection, Transfection, Standard Deviation, Negative Control

Journal: Experimental & Molecular Medicine

Article Title: Latrophilin-2 is a novel receptor of LRG1 that rescues vascular and neurological abnormalities and restores diabetic erectile function

doi: 10.1038/s12276-022-00773-5

Figure Lengend Snippet: a, b Tube formation assay (a) and transwell cell migration assays (b) using HUVECs treated with the indicated concentrations of the following proteins: PBS (negative control), LRG1 (1 μg/ml), TGF-β1 (5 ng/ml), LRG1 (1 μg/ml) + TGF-β1 (5 ng/ml), LRG1 (1 μg/ml) + anti-TGF-β1 Ab (10 μg/ml), and LRG1 (1 μg/ml) + TGF-β1 (5 ng/ml) + anti-TGF-β1 Ab (10 μg/ml). Left: Representative images. Scale bars, 200 µm. Right: Master junctions and migrated cells were quantified using ImageJ, and the results are presented as the means ± SEM ( n = 4). The relative ratio of the control group was defined as 1. c Cell surface binding of LRG1-YFP or YFP to HEK293T cells (top) and HUVECs (bottom) was assessed after treatment with LRG1-YFP (10 μg/ml) or YFP (10 μg/ml). Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 100 µm. d Schematic diagram of LRC-TriCEPS technique to identify a TGF-β-independent receptor of LRG1. e Volcano plots showing FDR-adjusted P values plotted against fold changes between samples comparing TriCEPS-coupled LRG1 or Transferrin with the glycine-quenched TriCEPS reagent control sample. The three proteins LPHN2, LEG3 and NDUA5 represent receptor candidates and were defined as those with an enrichment factor greater than 4 and an FDR-adjusted P value < 0.05. f LPHN2 expression in HEK293T cells and HUVECs after infection with scramble shRNA control (shCon, 5 × 10 4 TU/ml) or LPHN2 knockdown shRNA lentivirus (shNPHN2, 5 × 10 4 TU/ml) was analyzed by Western blotting. g Immunoprecipitation (IP) of LPHN2 in whole HUVEC lysates treated with or without LRG1 (1 μg/ml) for 10 min followed by immunoblot analysis to detect LRG1 and LPHN2. Data are the means ± SEM ( n = 3). h Cell surface binding of LRG1-YFP or YFP to control (shCon-expressing) or LPHN2-knockdown (shLPHN2-expressing) HEK293T cells (left) and HUVECs (right) after treatment with LRG1-YFP (10 μg/ml) or YFP (10 μg/ml). Nuclei were stained with DAPI (blue). Scale bar, 100 µm. * P < 0.05; ** P < 0.01 (Student’s t test). N.S., not significant.

Article Snippet: After the animals were anesthetized with intramuscular injections of ketamine (100 mg/kg) and xylazine (5 mg/kg), the scrambled control shRNA (shCon) lentivirus particles (Cat# SC-108080, Santa Cruz) or SMART vector mouse lentivirus containing shRNA targeting LPHN2 (shLPHN2; Cat# V3SM7603-234963095, Dharmacon) were administered by intramuscular injection at a dose of 1 × 10 5 transforming units (TU) per mouse.

Techniques: Tube Formation Assay, Migration, Negative Control, Control, Binding Assay, Labeling, Expressing, Infection, shRNA, Knockdown, Western Blot, Immunoprecipitation, Staining

Journal: Experimental & Molecular Medicine

Article Title: Latrophilin-2 is a novel receptor of LRG1 that rescues vascular and neurological abnormalities and restores diabetic erectile function

doi: 10.1038/s12276-022-00773-5

Figure Lengend Snippet: a , b Ex vivo mouse aortic ring assay ( a ) and mouse cavernosum endothelial cell (MCEC) sprouting assay ( b ) under normal-glucose (NG) or high-glucose (HG) conditions with treatment of PBS (negative control) or LRG1 (1 μg/ml) in the presence of lentivirus (shCon or shLPHN2; 5 × 10 4 TU/ml) or anti-TGF-β1 antibody (10 μg/ml) for 5 days. Sprouted microvessels were immunostained with the endothelial cell marker platelet/endothelial cell adhesion molecule-1 (PECAM-1) (red, a ). The dotted line indicates the sprouting endothelial cell range ( b ). Scale bars, 100 μm. The intensity of the area of microvessel sprouting from aortic rings (means ± SEM (n = 4) ( a ) and endothelial cell sprouting from cavernous tissue (means ± SEM ( n = 6) ( b ) were quantified (bottom). c and d βIII-Tubulin immunostaining in mouse MPG tissue ( c , top) and DRG tissue ( d , top) under normal glucose (NG) or high glucose (HG) conditions with treatment of PBS (negative control) or LRG1 (1 μg/ml) in the presence of lentivirus (shCon or shLPHN2; 5 × 10 4 TU/ml) or anti-TGF-β1 antibody (10 μg/ml) for 1 week. Scale bars, 100 µm. Quantification of βIII-tubulin–immunopositive neurite length in MPG ( c ) and DRG ( d ) tissue and (bottom, means ± SEM ( n = 4)). * P < 0.05; ** P < 0.01; *** P < 0.001 (Student’s t test). N.S., not significant. The relative ratio of the NG group was defined as 1.

Article Snippet: After the animals were anesthetized with intramuscular injections of ketamine (100 mg/kg) and xylazine (5 mg/kg), the scrambled control shRNA (shCon) lentivirus particles (Cat# SC-108080, Santa Cruz) or SMART vector mouse lentivirus containing shRNA targeting LPHN2 (shLPHN2; Cat# V3SM7603-234963095, Dharmacon) were administered by intramuscular injection at a dose of 1 × 10 5 transforming units (TU) per mouse.

Techniques: Ex Vivo, Aortic Ring Assay, Negative Control, Marker, Immunostaining

Journal: Experimental & Molecular Medicine

Article Title: Latrophilin-2 is a novel receptor of LRG1 that rescues vascular and neurological abnormalities and restores diabetic erectile function

doi: 10.1038/s12276-022-00773-5

Figure Lengend Snippet: a Penile tissue from normal mice was cross-sectioned for immunofluorescence staining. Representative LPHN2 (red), PECAM-1 (green, top left) and βIII-Tubulin (green, top right) staining. Nuclei were stained with DAPI (blue). Scale bars, 200 µm. Higher magnification images of the corpus cavernosum (CC; scale bars, 100 µm), cavernous artery (CA; scale bars, 25 µm), dorsal vein (DV; scale bars, 50 µm), dorsal artery (DA; scale bars, 25 µm) and dorsal nerve bundles (DNB; scale bars, 25 µm) (bottom). b Representative Western blots showing LPHN2 and LRG1 in mouse cavernous tissue from control and STZ-induced diabetic mice (top). Normalized band intensity values were quantified by ImageJ and are presented as the means ± SEM ( n = 4). c and d LPHN2 and LRG1 expression in CC ( c ) and DNB ( d ) tissue from control and STZ-induced diabetic mice (DM). Immunostaining for LPHN2 (red, left), LRG1 (red, middle) with PECAM-1 ( c , green) or βIII-Tubulin ( d , green). Scale bars, 100 μm. Immunopositive areas of LPHN2 and LRG1 in mouse CC (c) and DNB (d ) were quantified using ImageJ and are presented as the means ± SEM ( n = 6, right). e LPHN2 and LRG1 expression in human CC tissue from control and diabetic patients with erectile dysfunction (DM-ED). Immunostaining for LPHN2 (left, red) and LRG1 (middle, red). Nuclei were stained with DAPI (blue). Scale bars, 100 μm. Quantification of LPHN2- and LRG1-immunopositive areas in human CC using ImageJ (means ± SEM ( n = 6, right). f Representative intracavernous pressure (ICP) responses in control and STZ-induced diabetic mice at 2 weeks after repeated intracavernous injections of Fc (negative control, 5 µg/20 µl), LRG1-Fc (5 µg/20 µl), or LRG1-Fc (5 µg/20 µl) + anti-TGF-β1 antibodies (10 μg/ml) on Days 0 and 3 with scramble shRNA control (shCon) or LPHN2 knockdown shRNA (shLPHN2) conditions (5 × 10 5 TU/mouse). The cavernous nerve was stimulated with 5 V. The stimulus interval is indicated by a solid bar. The ratios of mean maximal ICP ( f , middle) and total ICP ( f , right) (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group ( n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001 (Student’s t test). N.S., not significant. The relative ratio of the control group was defined as 1.

Article Snippet: After the animals were anesthetized with intramuscular injections of ketamine (100 mg/kg) and xylazine (5 mg/kg), the scrambled control shRNA (shCon) lentivirus particles (Cat# SC-108080, Santa Cruz) or SMART vector mouse lentivirus containing shRNA targeting LPHN2 (shLPHN2; Cat# V3SM7603-234963095, Dharmacon) were administered by intramuscular injection at a dose of 1 × 10 5 transforming units (TU) per mouse.

Techniques: Immunofluorescence, Staining, Western Blot, Control, Expressing, Immunostaining, Negative Control, shRNA, Knockdown

Journal: Experimental & Molecular Medicine

Article Title: Latrophilin-2 is a novel receptor of LRG1 that rescues vascular and neurological abnormalities and restores diabetic erectile function

doi: 10.1038/s12276-022-00773-5

Figure Lengend Snippet: a Immunostaining of PECAM-1 (green) and neuron-glial antigen 2 (NG2, red) in cavernous tissue from control and STZ-induced diabetic mice at 2 weeks after repeated intracavernous injections of Fc (negative control, 5 µg/20 µl) or LRG1-Fc (5 µg/20 µl) in Days 0 and 3 under shCon or shLPHN2 conditions (5 × 10 5 TU/mouse) (left and middle). Scale bars, 100 μm. PECAM-1 and NG2 (+) areas per cavernosum were quantified using ImageJ (right, n = 5). b, d, e, f and g Immunostaining of p-eNOS ( b ), BrdU ( d ), Claudin-5 ( e ), oxidized-low-density lipoprotein (LDL, f ), TUNEL ( g ) (red) and PECAM-1 (green) in cavernous tissue from control and STZ-induced diabetic mice 2 weeks after repeated intracavernous injections of Fc or LRG1-Fc (5 µg/20 µl) on Days 0 and 3. Nuclei were stained with DAPI (blue, d and g ). Scale bars, 100 μm (scale bars, 50 μm for only d ). c Representative Western blots showing p-eNOS and eNOS in whole penile tissue from control and STZ-induced diabetic mice 2 weeks after repeated intracavernous injections of Fc (negative control, 5 µg/20 µl) or LRG1-Fc (5 µg/20 µl) on Days 0 and 3. h Quantification of p-eNOS by immunostaining and Western blotting (n = 4), the number of BrdU (+) endothelial cells per high-power field ( n = 4), claudin-5 and oxidized-LDL per cavernosum ( n = 4), and the number of TUNEL (+) apoptotic endothelial cells per high-power field ( n = 4). i and j Immunostaining of βIII-tubulin (green) and nNOS (red) in DNB tissue from control and STZ-induced diabetic mice 2 weeks after repeated intracavernous injections of Fc (negative control, 5 µg/20 µl) or LRG1-Fc (5 µg/20 µl) on Days 0 and 3 under shCon or shLPHN2 conditions (5 × 10 5 TU/mouse, only for i ). Nuclei were labeled with DAPI (blue). Scale bars, 25 µm for ( i ) and 100 μm for ( j ). βIII-tubulin or nNOS (+) areas in DNB tissue per high-power field were quantified using ImageJ (means ± SEM ( n = 6), right). * P < 0.05; ** P < 0.01; *** P < 0.001 (Student’s t test). N.S., not significant. The relative ratio of the shCon+Fc group or control group was defined as 1.

Article Snippet: After the animals were anesthetized with intramuscular injections of ketamine (100 mg/kg) and xylazine (5 mg/kg), the scrambled control shRNA (shCon) lentivirus particles (Cat# SC-108080, Santa Cruz) or SMART vector mouse lentivirus containing shRNA targeting LPHN2 (shLPHN2; Cat# V3SM7603-234963095, Dharmacon) were administered by intramuscular injection at a dose of 1 × 10 5 transforming units (TU) per mouse.

Techniques: Immunostaining, Control, Negative Control, TUNEL Assay, Staining, Western Blot, Labeling