primary endothelial cell culture medium  (iCell Bioscience Inc)

Journal: Signal Transduction and Targeted Therapy

Article Title: Anti-lymphangiogenesis for boosting drug accumulation in tumors

doi: 10.1038/s41392-024-01794-4

Figure Lengend Snippet: Effects of anlotinib and SAR131675 on the structure and function of tumor blood vessels, tumor stroma and IFP. a – e During the time and dosing window of anlotinib used in this experiment, tumor vessel normalization did not occur. Immunohistochemistry staining for endothelial cells (CD31, brown) (Scale bar, 100 μm), immunofluorescence staining for endothelial cells (CD31, red) and pericytes (NG2, green) (Scale bar, 80 μm), fluorescence images of Dylight @ 488-lectin-perfused (green) tumor blood vessels (CD31, red) (Scale bar, 40 μm), and representative images of HIF-1α (brown) immunohistochemical staining (Scale bar, 40 μm) in 4T1 tumor sections from mice treated with saline or anlotinib are shown in ( a ). Quantitative analysis of tumor vascular density ( b ), pericyte coverage ( c ; NG2 + CD31 + area percentage of the total CD31 + area), perfused vessels ( d ; lectin + CD31 + area percentage of the total CD31 + area), and HIF-1α area ( e ) as shown in ( a ) ( n = 9 or n = 12; images were from three mice per group). f – h SAR131675 did not influence the density and function of tumor blood vessels. Immunohistochemistry staining for endothelial cells (CD31, brown) and HIF-1α (brown) in 4T1 tumor sections from mice treated with saline or SAR131675 are shown in ( f ). Scale bar, 100 μm in the upper panels and 20 μm in the lower panels. Quantification of tumor vascular density ( g ) and HIF-1α area ( h ) as shown in ( f ) ( n = 9; images were from three mice per group). i – k Anlotinib and SAR131675 did not modulate tumor stroma. Histological studies with trichrome staining of collagen and immunohistochemical staining of fibronectin in tumor ( i ). Scale bar, 50 μm. Quantitative analysis of collagen ( j ) and fibronectin ( k ) ( n = 9; images were from three mice per group). l Tumor IFP of tumor-bearing mice treated with saline, anlotinib, or SAR131675 for 10 consecutive days ( n = 9). The data are shown as the mean ± s.d. ns no significance, *** p < 0.001

Article Snippet: CT26 and 4T1 cell lines were cultured in RPMI 1640 supplemented with 10% FBS, penicillin (100 units ml −1 ), and streptomycin (100 μg ml −1 ) at 37 °C in a humidified incubator with 5% CO 2 . hLECs was cultured in primary endothelial cell culture medium purchased from ScienCell (USA).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Fluorescence, Immunohistochemical staining, Saline

Journal: Molecular Medicine Reports

Article Title: SENP1 attenuates hypoxia‑reoxygenation injury in liver sinusoid endothelial cells by relying on the HIF‑1α signaling pathway

doi: 10.3892/mmr.2024.13188

Figure Lengend Snippet: Expression of SENP1 in liver sinusoidal endothelial cells following H-R. (A and B) Western blotting analysis. (C) Reverse transcription-quantitative polymerase chain reaction analysis. Data were presented as the mean ± SD (n=3); ***P<0.001, ****P<0.0001 vs. the control (normoxic) group. H-R, hypoxia-reoxygenation; SENP1, Sentrin/SUMO-specific protease 1.

Article Snippet: This company obtained LSECs from male C57BL/6 mice by digesting dissected liver tissue with elastase and collagenase and then culturing the cells at 37°C with 5% CO 2 in primary endothelial cell culture medium (iCell Bioscience, Inc.).

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: SENP1 attenuates hypoxia‑reoxygenation injury in liver sinusoid endothelial cells by relying on the HIF‑1α signaling pathway

doi: 10.3892/mmr.2024.13188

Figure Lengend Snippet: Effects of SENP1 expression downregulation and reactivation on the extent of fenestration damage and the reduction in the viability of H-R injured LSECs. (A) Representative scanning electron microscopy images showing fenestrae in each treatment group. (B) Scanning electron microscopy images showing the number of fenestrae in LSECs. (C) Cell Counting Kit-8 assay results. Data were presented as the mean ± SD (n=3); *P<0.05 vs. the control (normoxic) group; ****P<0.0001 vs. the H-R + si-NC group; ***P<0.001, ****P<0.0001 vs. the H-R + si-SENP1 group. H-R, hypoxia-reoxygenation; LSECs, liver sinusoidal endothelial cells; NC, negative control; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA.

Article Snippet: This company obtained LSECs from male C57BL/6 mice by digesting dissected liver tissue with elastase and collagenase and then culturing the cells at 37°C with 5% CO 2 in primary endothelial cell culture medium (iCell Bioscience, Inc.).

Techniques: Expressing, Electron Microscopy, Cell Counting, Control, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: SENP1 attenuates hypoxia‑reoxygenation injury in liver sinusoid endothelial cells by relying on the HIF‑1α signaling pathway

doi: 10.3892/mmr.2024.13188

Figure Lengend Snippet: Effects of SENP1 expression downregulation and reactivation on the apoptosis rate of H-R injured liver sinusoidal endothelial cells. (A and B) Flow cytometry plots. (C) Apoptosis rates of the normoxic and H-R groups. Data were presented as the mean ± SD (n=3); # P<0.05 vs. the normoxic group; ****P<0.0001 vs. the si-NC group; ***P<0.001 and *P<0.05 vs. the rescue group. H-R, hypoxia-reoxygenation; NC, negative control; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA.

Article Snippet: This company obtained LSECs from male C57BL/6 mice by digesting dissected liver tissue with elastase and collagenase and then culturing the cells at 37°C with 5% CO 2 in primary endothelial cell culture medium (iCell Bioscience, Inc.).

Techniques: Expressing, Flow Cytometry, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: SENP1 attenuates hypoxia‑reoxygenation injury in liver sinusoid endothelial cells by relying on the HIF‑1α signaling pathway

doi: 10.3892/mmr.2024.13188

Figure Lengend Snippet: SENP1, HIF-1α, HO-1, cleaved-caspase-3, Bax and Bcl-2 protein expression levels in SENP1-knockdown H-R-injured liver sinusoidal endothelial cells. (A) Representative western blots and ratio of SENP1, HIF-1α vs. GAPDH expression determined from the western blot images in the normoxia group. (B) Representative western blots and ratio of SENP1, HIF-1α in the H-R group. (C) Representative western blots and ratio of Bax and cleaved-caspase-3 in the normoxia group. (D) Representative western blots and ratio of Bax and cleaved-caspase-3 in the H-R group. (E) Representative western blots and ratio of Bcl-2 and HO-1 in the normoxia group. (F) Representative western blots and ratio of Bcl-2 and HO-1 in the H-R group. Date were shown as the mean ± SD (n=3); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the si-NC or si-SENP1 group. HIF-1α, hypoxia-inducible transcription factor-1α; HO-1, heme oxygenase; NC, negative control; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA; H-R, hypoxia-reoxygenation.

Article Snippet: This company obtained LSECs from male C57BL/6 mice by digesting dissected liver tissue with elastase and collagenase and then culturing the cells at 37°C with 5% CO 2 in primary endothelial cell culture medium (iCell Bioscience, Inc.).

Techniques: Expressing, Knockdown, Western Blot, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: SENP1 attenuates hypoxia‑reoxygenation injury in liver sinusoid endothelial cells by relying on the HIF‑1α signaling pathway

doi: 10.3892/mmr.2024.13188

Figure Lengend Snippet: VEGF, IL-6 and TNF-α protein levels in SENP1-knockdown hypoxia-reoxygenation-injured LSECs. (A) ELISA of VEGF expression in all LSEC groups. (B and C) ELISAs of IL-6 and TNF-α expression in all LSEC groups. Data were presented as the mean ± SD (n=5); **P<0.01, ***P<0.001, ****P<0.0001 vs. the si-NC or si-SENP1 groups; ## P<0.01, ### P<0.001 vs. the normoxic group. ELISA, enzyme-linked immunosorbent assay; LSECs, liver sinusoidal endothelial cells; NC, negative control; VEGF, vascular endothelial growth factor; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA.

Article Snippet: This company obtained LSECs from male C57BL/6 mice by digesting dissected liver tissue with elastase and collagenase and then culturing the cells at 37°C with 5% CO 2 in primary endothelial cell culture medium (iCell Bioscience, Inc.).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Expressing, Negative Control, Small Interfering RNA

Journal: BMC Complementary Medicine and Therapies

Article Title: Protective effects of the Terminalia bellirica tannin-induced Nrf2/HO-1 signaling pathway in rats with high-altitude pulmonary hypertension

doi: 10.1186/s12906-023-03981-2

Figure Lengend Snippet: TTR inhibits H 2 O 2 -induced endothelial cell injury in rat pulmonary arteries. ( A ) control group; ( B ) H 2 O 2 group; ( C ) TTR 20 µg; ( D ) TTR 40 µg; ( E ) TTR 80 µg; ( F ) results of the apoptosis analysis of PAECs

Article Snippet: Primary endothelial cell basal culture medium, cell growth factor, and penicillin‒streptomycin double antibiotic were obtained from iCell Bioscience Inc. Pancreatin (0.25%) was purchased from HyClone, USA.

Techniques: Control

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: RSPO2 inhibits VEGF-C-stimulated AKT and eNOS activation in LEC. (A–G) LEC were serum-starved in 0.5% FBS containing basal media MV2 for 16 h. Then, LEC were pretreated with RSPO2 (6 h), stimulated with VEGF-C for 15 min, and subjected to western blot analysis. (A and E) Representative western blot images are shown. (B–D and F) Bar diagrams represent averaged protein levels expressed as a ratio of phospho to total proteins, eNOS (B), AKT (C), ERK1/2 (D), and PKCδ (F) (n = 6–8). (G) Bar diagram represents the mean expression of VEGFR3 normalized with GAPDH (n = 6). (H–J) Control and LGR4-silenced LEC were used for this experiment. The details of treatment are same as in (A). (H) Representative western blot images are shown. (I and J) Bar diagrams represent averaged protein levels expressed as a ratio of phospho to total proteins, eNOS (I) (n = 4) and AKT (J) (n = 5). Statistical analyses were performed using one-way ANOVA (B–D, F, and G) and two-way ANOVA (I and J). Data represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. eNOS, endothelial nitric oxide synthase; LEC, lymphatic endothelial cells; RSPO2, R-spondin 2; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: Supplementation of nitric oxide by SNP abrogates RSPO2’s anti-lymphangiogenic activity. (A) Vehicle or RSPO2-pretreated LEC (6 h) were stimulated with VEGF-C for 1 h, washed, incubated with DAF-FM diacetate, and fluorescence determined (excitation/emission 495/515 nm). L-NAME pretreated cells were used as negative controls. Data are representative of three independent experiments performed in triplicate. (B) LEC were treated with vehicle or RSPO2 for 30 min, incubated with H2DCFDA and fluorescence analysed using flow cytometry. Representative histograms showing H2DCFDA fluorescence are shown. The X-axis is logarithmic. Bar diagram indicates mean fluorescence intensity in different groups (n = 4). (C) Human LEC were used to extract RNA and qRT-PCR was performed to determine levels of Nox1, Nox2, Nox4, and Nox5 expression. GAPDH was used as an internal control. Bar graph represents mRNA levels of different Nox isoforms in comparison to Nox2 (gene with the lowest expression). Data are representative of three independent experiments performed in triplicate. (D and E) LEC were pretreated with vehicle, SNP (1 µM, D) or EUK-134 (100 nM, E) for 30 min, then incubated with RSPO2 (6 h), stimulated with VEGF-C for 48 h and proliferation investigated using MTT assay. Data are representative of three to four independent experiments performed at least in triplicate. (F) LEC pretreated with vehicle or SNP (30 min) were incubated with RSPO2 for 6 h, trypsinized, and seeded in wells of Matrigel-coated plate in basal medium containing VEGF-C, VEGF-C + RSPO2, and VEGF-C + RSPO2 + SNP, and tube formation determined (6 h). Representative photomicrographs are shown. Scale bar 200 µm. Bar diagrams indicate tube length and number of branching points (n = 6–7). (G) Matrigel plugs mixed with VEGF-C, VEGF-C + RSPO2, or VEGF-C + RSPO2 + SNP were implanted subcutaneously in wild-type mice. Plugs were isolated after 2 weeks, sectioned and immunostained for LYVE-1. Bar diagram shows quantitative analysis of LYVE-1 positive area in implanted Matrigel plugs (n = 5–8). Statistical analyses were performed using one-way ANOVA (A and C–G) and two-tailed unpaired t-test (B). Data represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. L-NAME, L-NG-Nitro arginine methyl ester; LEC, lymphatic endothelial cells; LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1; RSPO2, R-spondin 2; SNP, sodium nitroprusside; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: Activity Assay, Incubation, Fluorescence, Flow Cytometry, Quantitative RT-PCR, Expressing, Control, Comparison, MTT Assay, Isolation, Two Tailed Test

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: RSPO2 suppresses lymphangiogenesis in vitro and in vivo. (A) Human LEC were pretreated with vehicle or RSPO2 (100 ng/mL) in basal media MV2 containing 0.5% FBS for 6 h, stimulated with VEGF-C (100 ng/mL) and proliferation investigated after 48 h using MTT assay. Data are representative of six independent experiments performed at least in triplicate. (B) LEC grown on coverslips were pretreated with vehicle or RSPO2 for 6 h and stimulated with VEGF-C for 24 h. Cells were fixed and immunostained for Ki67 (red). Nuclei and actin filaments were counterstained with To-Pro 3 (blue) and phalloidin (green), respectively. Images were captured from five to nine random fields. Representative images are shown. Scale bar 50 µm. Bar graph represents the mean number of Ki67 positive cells/field (n = 4). (C) Vehicle or RSPO2-pretreated LEC were seeded in upper chambers of transwell plate in basal medium containing VEGF-C ± RSPO2 and migration investigated after 12 h. Images of seven to nine randomly selected fields were acquired and number of migrated cells counted. Representative photomicrographs are shown. Scale bar 500 µm. (D) Bar graph represents the number of migrated cells (n = 5). (E–G) Vehicle or RSPO2-pretreated LEC were seeded in wells of a Matrigel-coated plate in basal medium containing VEGF-C ± RSPO2 and tube formation determined (6 h). Representative photomicrographs are shown. Scale bar 500 µm (E). Images of random fields were taken, and tube length (F) and number of branching points (G) quantified (n = 8). (H) Matrigel plugs mixed with either VEGF-C or VEGF-C + RSPO2 were implanted subcutaneously in wild-type mice. Plugs were isolated after 2 weeks, sectioned and immunostained for LYVE-1. (I) Quantitative analysis of LYVE-1 in implanted Matrigel plugs. Bar diagram represents mean LYVE-1 positive area (n = 6–8). Statistical analyses were performed using a two-tailed unpaired t-test. Data represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. LYVE-1, lymphatic vessel endothelial hyaluronan receptor-1; RSPO2, R-spondin 2; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: In Vitro, In Vivo, MTT Assay, Migration, Isolation, Two Tailed Test

Journal: Cardiovascular Research

Article Title: Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

doi: 10.1093/cvr/cvaa244

Figure Lengend Snippet: RSPO2 impairs Wnt-β-catenin signalling in VEGF-C-stimulated LEC. (A) HEK293 were treated with RSPO2 for 6 h and subjected to western blot analysis for LRP6 phosphorylation (left panel). LEC were serum-starved in 0.5% FBS containing basal media MV2 for 16 h. Then, LEC were pretreated with RSPO2 (6 h), stimulated with VEGF-C for 15 min, and subjected to western blot (right panel). (B) Bar diagram represents mean protein levels expressed as a ratio of phospho to total protein (n = 5). (C) LEC grown on coverslips were pretreated with vehicle or RSPO2 (6 h) and stimulated with VEGF-C for 15 min. Cells were fixed and immunostained for β-catenin (green). Nuclei were counterstained with Hoechst 33342 (red). At least five images of randomly chosen microscopic fields were captured. Representative images are shown. Scale bar 20 µm. Enlarged images; scale bar 10 µm. (D) Bar graph represents the mean co-localization coefficient for Hoechst 33342 and β-catenin (n = 14–17). (E) LEC were treated as indicated, nuclear-cytoplasmic fractionation conducted, and western blot experiments performed using nuclear and cytoplasmic fractions of LEC. GAPDH was used as cytoplasmic control. TBP and histone 3 were used to determine the purity of nuclear preparations. Bar diagram represents mean β-catenin levels in cytoplasmic and nuclear fractions (n = 4). Statistical analyses were performed using one-way ANOVA (B, D, and E). Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01. L-NAME, L-NG-Nitro arginine methyl ester; LEC, lymphatic endothelial cells; RSPO2, R-spondin 2; TBP, TATA-binding protein; VEGF, vascular endothelial growth factor.

Article Snippet: Cell culture Primary human dermal LEC (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium MV 2 (PromoCell) containing 5% heat-inactivated foetal bovine serum (FBS), 100 IU/mL of penicillin, 100 μg/mL streptomycin, and growth factors bullet kit provided by PromoCell.

Techniques: Western Blot, Phospho-proteomics, Fractionation, Control, Binding Assay