Journal: International Journal of Molecular Sciences

Article Title: Urokinase-Type Plasminogen Activator Receptor Regulates Prosurvival and Angiogenic Properties of Cardiac Mesenchymal Stromal Cells

doi: 10.3390/ijms242115554

Figure Lengend Snippet: Downregulation of uPAR reduced posttransplantation MPCs retention and vascularization after myocardial infarction. ( a , b ) Representative images of uPAR(−) ( a ) and uPAR(+) MPCs ( b ), labelled with the red fluorescent dye CM-DIL, in mice myocardium 72 h after transplantation. ( c ) Graphs quantifying the number of uPAR(+) and uPAR(−) MPCs retained in the myocardium 72 h after transplantation ( n = 4). ( d , e ) Representative images of vascularization of the periinfarction region of the hearts after transplantation of uPAR(−) ( d ) and uPAR(+) MPCs ( e ). Vessels were stained with antibodies against endothelial marker CD31 (red) and cell nuclei—with Dapi (blue). ( f ) Graphs quantifying the total number of CD31+ vessels in periinfarction zones (7 days after MI) after transplantation of uPAR(+) and uPAR(−) MPCs ( n = 4). Scale bar 100 mkm.

Article Snippet: Mouse neonatal cardiac endothelial cells were harvested by enzymatic homogenization of neonatal mouse hearts (2 days old) using a Neonatal Heart Dissociation Kit (Miltenyi Biotec, Gaithersburg, MD, USA) followed by immunomagnetic selection with a Neonatal Cardiac Endothelial Cell Isolation Kit (Miltenyi Biotec, USA).

Techniques: Transplantation Assay, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Urokinase-Type Plasminogen Activator Receptor Regulates Prosurvival and Angiogenic Properties of Cardiac Mesenchymal Stromal Cells

doi: 10.3390/ijms242115554

Figure Lengend Snippet: Knockdown of uPAR reduces secretion of proangiogenic factors and angiogenesis properties in vitro. ( a ) Representative images of tube-like structures on Matrigel (angiogenesis assay), formed by mouse endothelial cells in control medium ( a ) and after treatment with conditioned mediums of uPAR(−) ( b ) and uPAR(+) MPCs ( c ). ( d , e ) Graphs quantifying the total number and length of tube-like structures ( n = 3). Scale bar 200 mkm. ( f ) Heatmap of uPAR(+) and uPAR(−) MPCs secretome analyzed by Proteome Profiler Mouse Angiogenesis Array Kit.

Article Snippet: Mouse neonatal cardiac endothelial cells were harvested by enzymatic homogenization of neonatal mouse hearts (2 days old) using a Neonatal Heart Dissociation Kit (Miltenyi Biotec, Gaithersburg, MD, USA) followed by immunomagnetic selection with a Neonatal Cardiac Endothelial Cell Isolation Kit (Miltenyi Biotec, USA).

Techniques: Knockdown, In Vitro, Angiogenesis Assay, Control

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 2. mRNA expression levels of VEGF and MMP‑13 in the knee articular cartilage of mice among the Sham, Dmm and Dmm+Th groups (n=4 in each group). (A) Relative mRNA expression levels of VEGF in the medial articular cartilage. (B) Relative mRNA expression levels of MMP‑13 in the medial articular cartilage. The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; MMP‑13, matrix metalloproteinase‑13; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Expressing, Standard Deviation

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 3. Immunohistochemical analysis of VEGF expression in the knee articular cartilage of mice among the Sham, Dmm and Dmm+Th groups (n=4 in each group). (A) Immunohistochemistry staining of VEGF in the articular cartilage of the medial tibial plateau (magnification, x400, scale bar=100 µm). (B) Quantification of VEGF positive cells, based on the results of immunohistochemistry staining. The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry, Staining, Standard Deviation

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 5. ELISA analysis of serum VEGF concentration of mice among the Sham, Dmm and Dmm+Th groups (n=8 in each group). The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram of skin vasculature illustrates the organization of blood vessels in the skin layers. In the deep dermis, there are large-diameter blood vessels, including arteries and veins. The intermediate dermis contains arterioles and venules that branch from these arteries and veins, forming an intricate network. Lymphatic vessels are also located in the intermediate dermis. In the superficial dermis, capillaries form a highly branched network. (B) A representative maximum projection image from a whole-mount immunohistochemical analysis of mouse ear skin is presented. This analysis uses the pan-endothelial cell (EC) marker PECAM-1 to visualize skin vasculature from the deep to the superficial dermis. Different colors in the image indicate varying depths (Z-depth) within the dermis. Scale bar: 100 μm. (C) Representative whole-mount images of PECAM-1 + vasculature in the superficial, intermediate, and deep dermis of mouse ear skin are shown. Scale bars: 100 μm. (D) Experimental outline for generating diet-induced obesity (DIO) mice. Mice were fed either regular diet (10 Kcal % fat) or high-fat diet (60 Kcal % fat) from 6 weeks-of-age to 22 weeks-of-age. (E) Representative whole-mount images of the superficial dermal vasculature in the ear skin of control and DIO mice at 22 weeks-of-age, labeled with antibodies for the vascular smooth muscle cell marker αSMA (green or white), the vascular permeability marker PLVAP (MECA-32, red or white), along with PECAM-1 (blue or white) are presented. Scale bars: 100 μm. (F) Quantification of PLVAP + /PECAM-1 + capillaries in the superficial dermal vasculature from control and DIO mice is shown. The sample size is N = 6 in each group. (G) Representative transmission electron microscopy images of capillary ECs in the superficial dermis from control and DIO mice are presented. The dotted box regions in the left panels are magnified in the right panels. Fenestrae were observed only in DIO capillary ECs (arrowheads). Scale bars: 200 nm. (H) Quantification of endothelial fenestration in control and DIO capillary ECs is provided, showing both the number and percentage of non-fenestrated and fenestrated ECs. The sample sizes are as follows: N = 18 in control, N = 27 in DIO. Results are shown as the mean ± SEM. *p<0.05. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Immunohistochemical staining, Marker, Control, Labeling, Permeability, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram illustrates capillary ECs in the superficial dermal vasculature with or without a neutralizing anti-PLVAP antibody. In DIO capillary ECs, fenestrae form, which facilitates molecular leakage from blood to tissue (left). The anti-PLVAP antibody binds to the PLVAP protein, possibly obstructing the fenestrae and inhibiting molecular leakage (right). (B) Schematic diagram illustrating the administration of the anti-PLVAP antibody into DIO mice from 20 weeks-of-age to 22 weeks-of-age using an osmotic pump. (C) Illustration shows intravital imaging of mouse ear skin, along with representative images of dextran extravasation from superficial capillaries. Lectin (green) labels capillaries, and dextran (40 kDa, red) is visible inside the capillaries immediately after injection (t=0), gradually extravasating thereafter (t=10). Scale bars: 100 μm. (D) Representative time-course images show the extravasation kinetics of dextran (40 kDa) in control skin, DIO skin treated with saline, and DIO skin treated with the neutralizing anti-PLVAP antibody. Lectin labels capillaries (green) in the superficial dermis. Time-course rainbow color images show the intensity of dextran. The amount of extravasated dextran is quantified based on the intensity of the dextran signal outside the lectin + capillaries. Scale bars: 100 μm. (E) Changes in dextran (40 kDa) extravasation are shown for control skin (blue), DIO skin treated with saline (red), and DIO skin treated with the neutralizing anti-PLVAP antibody (green). (F) Quantitative measurements of dextran (40 kDa) extravasation at the 4-minute mark are shown. The sample sizes are as follows: N = 13 in control, N = 11 in DIO + Saline, N = 9 in DIO + PLVAP ab. Results are shown as the mean ± SEM. *p<0.05, **p<0.01. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Imaging, Injection, Control, Saline, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Schematic diagram illustrates the changes in vascular structure and sensory functions in the skin between control and DIO mice. In the skin of DIO mice, capillary ECs become fenestrated, leading to increased vascular permeability. Additionally, DIO mice exhibit enhanced pain behavior and sensory hypersensitivity . (B) Illustration shows the implantation of an osmotic pump in sensory neuron-specific Pirt-GCaMP3 calcium reporter mice. This pump is used to administer saline, the IgG control, or the neutralizing anti-PLVAP antibody. The sample sizes are as follows: N = 6 in Pirt-GCaMP3 mice on a control diet (control), N = 8 in Pirt-GCaMP3 mice with DIO receiving saline (DIO + Saline), N = 5 in Pirt-GCaMP3 mice with DIO receiving IgG control (DIO + IgG control), N = 8 in Pirt-GCaMP3 mice with DIO receiving the anti-PLVAP antibody (DIO + PLVAP Ab). (C) Illustrations depict the capsaicin-mediated acute pain behavior assay (left) and ex vivo Ca 2+ imaging of peripheral terminals of nociceptive neurons located in the epidermis of the ear skin (right). (D) Total forelimb wiping responses following capsaicin application are shown for control, DIO + Saline, DIO + IgG control, and DIO + PLVAP Ab. (E) Quantification of Ca 2+ responses within the ear skin of control mice, DIO mice with saline, DIO mice with the IgG, and DIO mice with the neutralizing anti-PLVAP antibody is shown. The Ca 2+ transients were normalized by the baseline Ca 2+ transient (ΔF/F 0 ). (F) The integrated Ca 2+ transient (ΔF/F0) was calculated as the area under the curve (AUC). Results are shown as the mean ± SEM. *p<0.05, ***p<0.001. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Control, Permeability, Saline, Behavioral Assay, Ex Vivo, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: OBESITY-INDUCED ENDOTHELIAL FENESTRATION AND CAPILLARY LEAKAGE CONTRIBUTE TO INCREASED PAIN SENSATION

doi: 10.64898/2026.03.13.711502

Figure Lengend Snippet: (A) Representative section immunohistochemical images of ear skin from control mice, DIO mice treated with saline, and DIO mice treated with the neutralizing anti-PLVAP antibody are presented. This assay uses the antibodies for FOXO1 (green), the keratinocyte marker K14 (red), along with the nuclear marker TOPRO3 (blue). Each inset displays the pattern of FOXO1 expression in a single keratinocyte. Dashed lines indicate the boundary between the epidermis and the dermis. “Epi” indicates the epidermis; “D” indicates the dermis. Scale bars: 20 μm. (B) Quantification of nuclear FOXO1 expression in keratinocytes is provided. The percentages of nuclear FOXO1 expression within the total FOXO1 expression in keratinocytes are presented. The sample sizes are as follows: N = 5 in control, N = 5 in DIO + Saline, N = 7 in DIO + PLVAP Ab. (C) Representative X-gal staining images of ear skin from NGF-LacZ control mice, DIO mice with saline, and DIO mice with the neutralizing anti-PLVAP antibody (blue) are presented. Dashed lines indicate the boundary between the epidermis and the dermis. Scale bars: 50 μm. (D) Quantification of the LacZ-positive area in the epidermis is provided. The sample sizes are as follows: N = 9 in control, N = 15 in DIO + Saline, N = 9 in DIO + PLVAP Ab. (E) Graphical summary illustrates how vascular hyperpermeability leads to sensory hypersensitivity in DIO skin. Increased permeability in the superficial dermal capillaries facilitates the diffusion of insulin into the epidermis, activating insulin signaling in epidermal keratinocytes. This activation leads to NGF upregulation in these keratinocytes, which in turn promotes sensory hypersensitivity in DIO skin. A neutralizing anti-PLVAP antibody reduces the diffusion of insulin, thereby decreasing NGF expression in the epidermal keratinocytes and alleviating sensory hypersensitivity. Results are shown as the mean ± SEM. *p<0.05, **p<0.01. P values were determined by the parametric two-tailed t test. The schematic diagrams and graphic summary were partially created with BioRender.com .

Article Snippet: A neutralizing anti-PLVAP antibody (clone MECA-32, BioXCell, BE0200) or a rat IgG2a isotype control (BioXCell, BE0089) was diluted to a concentration of 2.11 mg/ml in a 0.9% sodium chloride solution (Millipore Sigma, S8776).

Techniques: Immunohistochemical staining, Control, Saline, Marker, Expressing, Staining, Permeability, Diffusion-based Assay, Activation Assay, Two Tailed Test

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Coronary Microvascular Dysfunction Is Associated With Augmented Lysosomal Signaling in Hypercholesterolemic Mice

doi: 10.1161/JAHA.124.037460

Figure Lengend Snippet: MCECs were cultured in low glucose DMEM with 5% FBS and treated with 7‐ketocholesterol for the indicated times. A and D , Representative immunofluorescence images and quantification show mitochondrial superoxide levels. Representative immunofluorescence images and quantification show the expression of proinflammatory proteins VCAM‐1 ( B and E ) and CCL2 ( C and F ). G , Representative immunofluorescence images and quantification show the nuclear TFEB positive percentage. Nuclei were stained with DAPI. H , Representative immunoblots and summarized data show the effects of 7‐ketocholesterol on the protein expression levels of microtubule‐associated proteins light chain 3‐II. I , Real‐time reverse transcription polymerase chain reaction analyses of TFEB, LAMP‐1, LAMP‐2A, beclin‐1, microtubule‐associated proteins light chain 3, and p62/SQSTM1 mRNA levels after treatment with 0 or 40 μM 7‐ketocholesterol for 24 hour. Scale bar=20 μm. * vs 0, P <0.05 (n=4–5). CCL2 indicates C‐C motif chemokine ligand 2; LAMP‐1, lysosomal‐associated membrane protein; MCECs, mouse cardiac endothelial cells; TFEB, transcriptional factor EB; and VCAM‐1, vascular cell adhesion molecule 1.

Article Snippet: MCECs (Cedarlane, CLU510) were cultured in low glucose DMEM with 5% FBS, 1% penicillin/streptomycin, and 1 mmol/L of HEPES at 37 °C with 5% CO 2 .

Techniques: Cell Culture, Immunofluorescence, Expressing, Staining, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Membrane

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Coronary Microvascular Dysfunction Is Associated With Augmented Lysosomal Signaling in Hypercholesterolemic Mice

doi: 10.1161/JAHA.124.037460

Figure Lengend Snippet: MCECs were cultured and treated in low glucose DMEM with 5% FBS, pretreated with or without 50 nM BAF for 1 hour, and then cotreated with or without 40 μM of 7‐ketocholesterol for 6 hour. A , Representative immunofluorescence images and quantification show the nuclear TFEB‐positive percentage. Nuclei were stained with DAPI. B through G , MCECs are treated in low‐glucose DMEM with 1% FBS for 2 hour before pretreatment with or without 50 nM of BAF for 1 hour, and then the cells are cotreated with or without 40 μM of 7‐ketocholesterol for 24 hour. B , Representative images of VCAM‐1 and summarized data. C through G , Representative images of FLICA/PI staining and summarized data. H , Cell numbers were detected by using CCK8 kit. Scale bar=20 μm. * vs 0, # vs BAF or 7‐ketocholesterol, P <0.05 (n=4). BAF indicates bafilomycin A1; CCK8, Cell‐Counting Kit 8; MCECs, mouse cardiac endothelial cells; PI, propidium iodide; TFEB, transcriptional factor EB; and VCAM‐1, vascular cell adhesion molecule 1.

Article Snippet: MCECs (Cedarlane, CLU510) were cultured in low glucose DMEM with 5% FBS, 1% penicillin/streptomycin, and 1 mmol/L of HEPES at 37 °C with 5% CO 2 .

Techniques: Cell Culture, Immunofluorescence, Staining, Cell Counting

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Coronary Microvascular Dysfunction Is Associated With Augmented Lysosomal Signaling in Hypercholesterolemic Mice

doi: 10.1161/JAHA.124.037460

Figure Lengend Snippet: MCECs were cultured in low glucose DMEM with 5% FBS, then treated with EZE with or without 7‐ketocholesterol for the indicated time. A , Representative immunofluorescence images and quantification show the effect of ezetimibe on nuclear TFEB positive percentage. B , Representative immunofluorescence images and quantification show the effect of ezetimibe and 7‐ketocholesterol on nuclear TFEB positive percentage. Representative immunofluorescence images and quantification of mitochondrial superoxide ( C ), proinflammatory proteins vascular cell adhesion molecule 1 (VCAM‐1) ( D ) and CCL2 ( E ), and monocyte adhesion ( F ). Scale bar=20 μm. * vs 0, # vs 7‐keto, P <0.05 (n=4). CCL2 indicates C‐C motif chemokine ligand 2; EZE, ezetimibe; MCECs, mouse cardiac endothelial cells; TFEB, transcriptional factor EB; and VCAM‐1, vascular cell adhesion molecule 1.

Article Snippet: MCECs (Cedarlane, CLU510) were cultured in low glucose DMEM with 5% FBS, 1% penicillin/streptomycin, and 1 mmol/L of HEPES at 37 °C with 5% CO 2 .

Techniques: Cell Culture, Immunofluorescence

Journal: bioRxiv

Article Title: Immunoregulatory subtype of dermal lymphatic endothelial cells at capillary terminals drives lymphatic malformations

doi: 10.1101/2022.05.22.492950

Figure Lengend Snippet: ( A-E ) Quantification of the CD45 + area ( A , B ) and F4/80 + area ( C - E ) in the ear skin showing increase in Pik3ca H1047R ;Vegfr3-CreER T2 ( A, C, D ) but not in Pik3ca H1047R ;Vegfr1-CreER T2 ( B, C, E ) mice. Data represent mean (CD45: n =4-6 images from n =3-5 mice per genotype; F4/80: n =3-7 images from n =2-3 mice per genotype) ± s.e.m. Representative binary images are shown below the graphs. ( F, G ) Flow cytometry analysis of innate ( F ) and adaptive ( G ) immune cells in the ear skin of 4-OHT-treated 5-week-old and 10-week-old Pik3ca H1047R ; Vegfr3-CreER T2 mice and littermate controls. Mac, macrophage; Mono, monocyte; DC, dendritic cell; Neu, neutrophil; NK, natural killer cell. Data represent relative cell frequency (of live cells) relative to the control ( n =3-9 mice) ± s.e.m. ( H ) Multiplex ELISA analysis of pro-inflammatory cytokines and chemokines associated with recruitment and/or activation of myeloid cells in whole ear skin lysates from Pik3ca H1047R ;Vegfr3-CreER T2 mice and littermate controls. Data represent mean protein levels relative to control ( n =6-11 mice) ± s.e.m. ( I ) qRT-PCR analysis (left) and ELISA analysis (right) of Vegfc /VEGF-C levels in the ear skin lysates of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr3-CreER T2 and littermate control mice. qRT-PCR data represent mean relative expression (normalized to Hprt ; n =7-10 mice) ± s.e.m. Transcript and protein levels are presented relative to controls. ( J ) qRT-PCR analysis of Vegfc in CD45 + Cd11b + F4/80 + macrophages and CD45 + Cd11b + F4/80 + Ly6C + monocytes, FACS-sorted from the ear skin of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr3-CreER T2 and littermate control mice. qRT-PCR data represent mean relative expression (normalized to Hprt ; n =3-5 mice) ± s.e.m. Transcript and protein levels are presented relative to macrophages/monocytes in control mice. p -value in (A, B, D-I) obtained using Two-tailed unpaired Student’s t-test. Scale bar: 100 μm (A-E).

Article Snippet: Mouse Vascular Endothelial Cell Growth Factor C (VEGF-C) ELISA Kit from CUSABIO (CSB-E07361m) was used for detection of VEGF-C protein concentration.

Techniques: Flow Cytometry, Control, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Immunoregulatory subtype of dermal lymphatic endothelial cells at capillary terminals drives lymphatic malformations

doi: 10.1101/2022.05.22.492950

Figure Lengend Snippet: ( A ) Flow cytometry analysis of the number of CD45 + CD11b + F4/80 + macrophages in the ear skin of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr3-CreER T2 ( n =5) and control ( n =3) mice. Data represent mean cell number per gram tissue ± s.e.m. p -value, Mann-Whitney U Test. ( B ) Flow cytometry analysis of the frequency of CD45 + CD11b + F4/80 + antigen-presenting myeloid cells in the ear skin of 4-OHT-treated 5-week-old Pik3ca H1047R ;Vegfr1-CreER T2 ( n =6) and control ( n =5) mice. Data represent relative cell frequency (of live cells) relative to the control ± s.e.m. p -value obtained using Two-tailed unpaired Student’s t-test. ( C ) Flow cytometry analysis of innate (left) and adaptive (right) immune cells in the ear skin of 4-OHT-treated 10-week-old Pik3ca H1047R ; Vegfr1-CreER T2 mice and littermate controls. Mac, macrophage; Mono, monocyte; DC, dendritic cell; Neu, neutrophil; NK, natural killer cell. Data represent relative cell frequency (of live cells) relative to the control ( n =5-8 mice for innate panel, n=7-9 mice for adaptive panel) ± s.e.m. p -value obtained using Two-tailed unpaired Student’s t-test. ( D, E ) Multiplex ELISA analysis of pro-inflammatory cytokines and chemokines associated with recruitment and/or activation of myeloid cells or T-cells and B-cells in whole ear skin lysates from Pik3ca H1047R ;Vegfr1-CreER T2 ( D ) and Pik3ca H1047R ;Vegfr3-CreER T2 ( E ) mice, and respective littermate controls. ( F ) Similar analysis of TNFα and INFγ in blood serum of Pik3ca H1047R ;Vegfr3-CreER T2 mice. Data in ( D-E ) represent mean protein levels ( n =3-9 mice) ± s.e.m.

Article Snippet: Mouse Vascular Endothelial Cell Growth Factor C (VEGF-C) ELISA Kit from CUSABIO (CSB-E07361m) was used for detection of VEGF-C protein concentration.

Techniques: Flow Cytometry, Control, MANN-WHITNEY, Two Tailed Test, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Activation Assay