Journal: Journal of Biological Chemistry

Article Title: Critical Role for GATA3 in Mediating Tie2 Expression and Function in Large Vessel Endothelial Cells

doi: 10.1074/jbc.m109.041145

Figure Lengend Snippet: FIGURE 6. siRNA-mediated knockdown of GATA3 attenuates Ang-1-induced AKT phosphorylation, migration, tube formation, and cell survival in primary human endothelial cells. A, left, HUVEC were transfected with si-control (lanes 1 and 3) or si-GATA3 (lanes 2 and 4) for 2 days, serum-starved for 18 h, and treated in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of Ang-1 for 10 min. Samples were immunoprecipitated with anti-AKT antibody and immunoblotted with phospho-specific AKT antibody. The membrane was stripped and re-probed for total AKT. Right, quantitation of phospho-AKT levels relative to si-control-transfected cells treated in the absence or presence of Ang-1. Data are expressed as mean S.D. from three independent experiments. #, p 0.0001; *, p 0.0001 compared with without Ang-1 control and control plus Ang-1, respectively N.S., nonsignificant. B, modified Boyden chamber assay of HUVEC transfected with si-control (Control) or si-GATA3 (GATA3) and treated in the presence or absence of Ang-1 or VEGF. Shown are the mean S.D. of migrated cells quantified by cell image analyzer in three independent experiments. #, p 0.01; *, p 0.001 compared with minus control and control plus Ang-1, respectively. N.S., nonsignificant. C, tube formation assay of si-control- or si-GATA3-transfected HUVEC plated on collagen gel and overlaid with fibroblast-containing collagen with or without Ang-1. Capillary-like tube morphology was observed under the fluorescent microscopy. Bar, 500 m. Quanti- fication of the tube length (bar graph) was calculated using the cell image analyzer from four arbitral optical images per experiment. Data are expressed as mean S.D. from three independent experiments. #, p 0.005; *, p 0.001 compared with without Ang-1 control and control plus Ang-1, respectively. N.S., nonsignificant. D, HUVEC were incubated with EBM-2 0.5% FBS for 18 h and then further incubated with Dulbecco’s modified Eagle’s medium 0.5% FBS (Starvation) for 24 h in the presence or absence of Ang-1. Cells were harvested and stained with annexin V-fluorescein isothiocyanate. Count of annexin V-positive cells were performed by flow cytometry. Data are presented as mean S.D. from six independent experiments. *, p 0.0001 compared with starvation alone. N.S., nonsignificant.

Article Snippet: Human skin fibroblast (Clonetics) and COS-7 cells (ATCC CRL-1651) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS).

Techniques: Knockdown, Phospho-proteomics, Migration, Transfection, Control, Immunoprecipitation, Membrane, Quantitation Assay, Modification, Boyden Chamber Assay, Tube Formation Assay, Microscopy, Incubation, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Dual Targeting of HIF-1α and DLL4 by Isoxanthohumol Potentiates Immune Checkpoint Blockade

doi: 10.3390/ijms27031576

Figure Lengend Snippet: Screening of natural flavanone derivatives for dual inhibition of HIF-1α and DLL4 promoter activities. ( A , B ) Cytotoxicity assessment of natural flavanone derivatives in HEK293 ( A ) and EA.hy926 ( B ) cells. Cells were treated with 10 µM of each natural flavanone derivative for 24 h, and cell viability was determined by MTT assay. ( C ) Inhibitory effect of flavanone derivatives on HRE-luciferase activity. HEK293 cells were co-transfected with pGL3-HRE and pRL-SV40 Renilla luciferase plasmid for 24 h, followed by treatment with 10 µM of natural flavanone derivatives or STP (used as a positive control) under hypoxic conditions for an additional 24 h. HRE-luciferase activity was measured using a dual-luciferase assay. ( D ) Inhibitory effect of flavanone derivatives on DLL4 promoter activity. EA.hy926 cells were co-transfected with pGL3-DLL4 and pRL-SV40 Renilla luciferase plasmids for 24 h, pre-incubated with 10 µM of natural flavanone derivatives for 1 h, and subsequently incubated for 24 h in the presence or absence of VEGF-A (10 ng/mL). DLL4-luciferase activity was measured using a dual-luciferase assay. Data are presented as the mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control ( A , B ), hypoxic control ( C ), or VEGF-treated control ( D ).

Article Snippet: HEK293 human embryonic kidney epithelial cells (KCLB, Seoul, Republic of Korea), EA.hy926 endothelial cells (ATCC, Manassas, VA, USA), and LLC cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% antibiotics (penicillin 100 units/mL and streptomycin 100 mg/mL; Invitrogen, Carlsbad, CA, USA).

Techniques: Inhibition, MTT Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Positive Control, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Targeting of HIF-1α and DLL4 by Isoxanthohumol Potentiates Immune Checkpoint Blockade

doi: 10.3390/ijms27031576

Figure Lengend Snippet: Effect of IXN on HIF-1α and DLL4. ( A , B ) Dual-luciferase assay confirming the inhibitory effects of IXN. Half-maximal inhibitory concentration (IC 50 ) values were calculated using GraphPad Prism software (version 8.0). The red dotted lines indicate the IC 50 values. ( C – F ) HEK293, LLC, and A549 cells were treated with IXN (0–10 µM) under hypoxic conditions for 16 h. HIF-1α protein expression levels were analyzed by Western blot and quantified using ImageJ software (version 1.54p). ( G – I ) EA.hy926 cells were treated with IXN (0–10 µM) in the presence of VEGF-A (10 ng/mL) for 24 h. DLL4 and NICD protein levels were analyzed by Western blot and quantified using ImageJ. Data are presented as the mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the hypoxic control ( D – F ) or VEGF-treated control ( H , I ).

Article Snippet: HEK293 human embryonic kidney epithelial cells (KCLB, Seoul, Republic of Korea), EA.hy926 endothelial cells (ATCC, Manassas, VA, USA), and LLC cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% antibiotics (penicillin 100 units/mL and streptomycin 100 mg/mL; Invitrogen, Carlsbad, CA, USA).

Techniques: Luciferase, Concentration Assay, Software, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Targeting of HIF-1α and DLL4 by Isoxanthohumol Potentiates Immune Checkpoint Blockade

doi: 10.3390/ijms27031576

Figure Lengend Snippet: Effect of IXN on HIF-1α/VEGF and DLL4/NOTCH1 signaling. ( A ) HEK293 cells were treated with STP (10 µM, positive control) or IXN (10 µM) under hypoxic conditions for 24 h, and HIF-1α protein levels were determined by Western blot. ( B ) EA.hy926 cells were treated with STP (10 µM) or IXN (10 µM) and incubated with or without VEGF-A (10 ng/mL) for 24 h. DLL4 expression was assessed by Western blotting. ( C ) HEK293 cells were treated with STP (10 µM) or IXN (10 µM) under hypoxia for 24 h, and VEGFA mRNA expression was quantified by RT-qPCR. ( D ) EA.hy926 cells were treated with STP (10 µM) or IXN (10 µM) and incubated with or without VEGF-A (10 ng/mL) for 24 h. DLL4 mRNA expression was quantified by RT-qPCR. ( E ) EA.hy926 cell proliferation was evaluated using a BrdU proliferation assay after treatment with STP (10 µM) or IXN (10 µM) in the presence or absence of VEGF-A (10 ng/mL) for 24 h. ( F , G ) Wound healing assay: EA.hy926 monolayers were scratched, treated with STP (10 µM) or IXN (10 µM) for 1 h, followed by VEGF-A (10 ng/mL) stimulation for 24 h. Representative images ( F ) and quantitative analysis of wound closure ( G ) are shown. Yellow dashed lines indicate the wound edges at 0 h (upper line) and 24 h (lower line). Scale bar: 100 µm. ( H – J ) Tube formation assay: EA.hy926 cells were seeded on Matrigel-coated wells with VEGF-A (10 ng/mL) and treated with STP (10 µM) or IXN (10 µM) for 24 h. Representative images were acquired using a light microscope ( H ). Quantitative analysis of total tube length ( I ) and branching points ( J ) was performed using ImageJ software (version 1.54p). Scale bar: 100 µm. ( K , L ) Wound healing assay: EA.hy926 cells were scratched and incubated with either fresh medium or A549-derived conditioned medium (CM) for 24 h. Representative images ( K ) and quantification of wound closure ( L ) are shown. Yellow dashed lines indicate the wound edges at 0 h (upper line) and 24 h (lower line). Scale bar: 100 μm. ( M – O ) Tube formation assay: EA.hy926 cells were seeded on Matrigel and incubated with fresh medium or CM for 8 h. Representative images ( M ) and quantification of total tube length ( N ) and branching points ( O ) are shown. Scale bar: 100 μm. CM were obtained from A549 cells cultured under hypoxic conditions (1% O 2 ) with vehicle (DMSO, CM-Vehicle), STP (10 μM, CM-STP), or IXN (10 μM, CM-IXN) for 24 h. Data are presented as the mean ± SD from three independent experiments. *** p < 0.001, and **** p < 0.0001 vs. hypoxic control ( C ), VEGF-treated control ( D , E , G , I , J ), or CM-Vehicle ( L , N , O ).

Article Snippet: HEK293 human embryonic kidney epithelial cells (KCLB, Seoul, Republic of Korea), EA.hy926 endothelial cells (ATCC, Manassas, VA, USA), and LLC cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% antibiotics (penicillin 100 units/mL and streptomycin 100 mg/mL; Invitrogen, Carlsbad, CA, USA).

Techniques: Positive Control, Western Blot, Incubation, Expressing, Quantitative RT-PCR, Proliferation Assay, Wound Healing Assay, Tube Formation Assay, Light Microscopy, Software, Derivative Assay, Cell Culture, Control

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: PSMA was detected in HUVECs cultured with the conditioned medium derived from LNCaP cells. (A) Confocal images of prostate cancer cells (PC3, DU145, LNCaP cells) cultured in condition 1 or condition 2. The scheme of each condition by which each conditioned medium (CM) was prepared is shown on the right. To prepare the CM, 1.5 × 10 5 cells of prostate cancer cells were seeded on 6‐well plastic dishes (condition 1) or collagen I gels (condition 2). Three days later, the media were replaced with fresh media. The cells were then incubated for another 3 days, then the media were collected as CM. Bars: 100 µm. (B, C) Confocal images of HUVECs cultured with the CM derived from LNCaP cells (B) or PC3 and DU145 cells (C). The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. (D) Western blots of HUVEC lysates cultured with the CM derived from prostate cancer cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. The lysates from LNCaP cells were used as a positive control of PSMA expression. (E) The mRNA expression of PSMA in HUVECs cultured with the CM derived from LNCaP cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Data are mean ± SEM from three independent experiments. ** p < .01. HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Cell Culture, Derivative Assay, Incubation, Concentration Assay, Immunofluorescence, Staining, Western Blot, Positive Control, Expressing

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: Fractionation of the CM derived from LNCaP cells. (A) Confocal images of HUVECs cultured with each fraction of CM derived from LNCaP cells. Each fraction was diluted to half of its concentration with EBM‐2 or suspended with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. The PSMA‐positive HUVECs were shown by arrowheads. Bars: 100 µm. The representative images from three independent experiments were shown. (B) Confocal images of HUVECs cultured with 10,000 g pellet fractions of CM derived from LNCaP cells. The 10,000 g pellet fraction was labeled with a membrane marker dye, before the suspension in EBM‐2 medium. The labeled 10,000 g pellet was cultured with HUVECs for 6 h, and cells were subjected to immunofluorescence staining for PSMA. Bars: 10 µm (left) and 2 µm (right; magnified images of squares in left images). The representative images from three independent experiments were shown. (C) Western blots of 10,000 g pellet fraction of CM derived from LNCaP cells. The lysates from HUVECs and LNCaP cells were used as negative and positive controls of PSMA expression, respectively. The representative blot data from three independent experiments were shown. (D) Confocal images of HUVECs cultured with the CM derived from PC3 cells that stably express Myc‐PSMA or PSMA‐myc. The CM was diluted in half with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. The representative images from three independent experiments were shown. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Fractionation, Derivative Assay, Cell Culture, Concentration Assay, Immunofluorescence, Staining, Labeling, Marker, Western Blot, Expressing, Stable Transfection

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: The tube formation assay of HUVECs cultured with 10,000 g pellet fraction of CM derived from prostate cancer cells. (A) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from LNCaP cells for 6 h, and packed on collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. To examine the PSMA dependency, the CM was prepared from LNCaP cells depleted of PSMA. Bars: 100 µm. (B) The quantitation of (A). Total tube lengths from three independent experiments were measured and normalized to those of cells cultured with normal EBM‐2. Data are the means ± SEM . * p < .05; n.s., not significant. (C) Western blots of LNCaP cell lysates, 72 h posttransfection with the indicated siRNAs. (D) Confocal images of PC3 cells stably expressing Myc‐PSMA or PSMA‐myc. Bars: 100 µm. (E) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from PC3 cells for 6 h, and packed in collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. Bars: 100 µm. (F) The quantitation of (E). Total tube lengths from three independent experiments were measured and normalized to those of PC3 (parental). Data are the means ± SEM . * p < .05; ** p < .01. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Tube Formation Assay, Cell Culture, Derivative Assay, Staining, Quantitation Assay, Western Blot, Stable Transfection, Expressing

Journal: Cancer Biology & Therapy

Article Title: Lenvatinib potentiates the antitumor efficacy of combined radiotherapy and PD-L1 blockade in lung adenocarcinoma

doi: 10.1080/15384047.2025.2610526

Figure Lengend Snippet: Radiation induces dual adaptive resistance pathways, and lenvatinib counteracts RT-induced PD-L1 upregulation. (A-B) Western blot analysis (A) and quantification (B) of PD-L1 protein expression in HCC827 cells 24 hours after gradient X-ray irradiation (0-20 Gy, n = 3). (C-D) Western blot analysis (C) and quantification (D) of PD-L1 expression in HCC827 cells 24 hours after 8 Gy irradiation, treated with lenvatinib (10 µM) and/or anti-PD-L1 antibody (40 µg/mL, n = 3). (E-F) Representative IF images (E) and quantification (F) of PD-L1 surface expression (MFI) in A549 cells treated with 8 Gy RT and Lenvatinib (10 µM, n = 3). Scale bar = 50 µm. (G) ELISA analysis of secreted VEGF-A protein in the supernatants of HCC827 cells at indicated time points (0-24 h) after 8 Gy irradiation ( n = 3). (H) RT-qPCR analysis of VEGFA mRNA expression in HCC827 cells 24 hours after 8 Gy irradiation ( n = 3). (I-J) Representative IF images (I) and quantification (J) of VEGFR2 expression in HUVECs ( n = 3). Scale bar = 50 µm. Data are presented as mean ± SEM. Statistical analysis was performed using Student's t-test, Mann-Whitney U test, or One-way ANOVA. (ns: P ≥ 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

Article Snippet: Proteins (40 μg) were separated via SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk in TBST (0.05% Tween-20) for one hour at RT, and incubated overnight at 4 °C with primary antibodies: VEGFR2 (D5B1 Rabbit mAb, 1:1,000, CST #9698), PD-L1 (1:1,000, Abcam #ab213524), and GAPDH (D16H11 XP® Rabbit mAb, 1:1,000, CST #5174).

Techniques: Western Blot, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancer Biology & Therapy

Article Title: Lenvatinib potentiates the antitumor efficacy of combined radiotherapy and PD-L1 blockade in lung adenocarcinoma

doi: 10.1080/15384047.2025.2610526

Figure Lengend Snippet: Lenvatinib inhibits tumour-induced endothelial activation via VEGFR2 blockade and angiogenic disruption. (A, C) Western blot analysis of VEGFR2 expression in HUVECs treated with increasing concentrations of lenvatinib (0-40 μM) for 24 hours ( n = 3). (B, D) Representative IF images (B) and quantification (D) of VEGFR2 expression (MFI) in HUVECs treated with lenvatinib (10 µM, n = 3). Scale bar = 50 µm. (E) Schematic of the in vitro co-culture model, with HCC827 cells cultured on a 0.4-μm transwell insert above a monolayer of HUVECs (Created with BioRender.com). (F) CCK-8 viability assay of HUVECs cultured alone (monoculture) or co-cultured with HCC827 cells, with or without lenvatinib (10 µM) for 24 hours ( n = 6). (G-I) In vitro tube formation assay using HUVECs cultured in conditioned medium from irradiated (8 Gy) HCC827 cells, which were pretreated with or without lenvatinib. (G) Representative images of Calcein AM-stained HUVEC networks (4-8 h). (H) Quantification of the number of junctions. (I) Quantification of total vascular length ( n = 3). Scale bar = 50 µm. Data are presented as mean ± SEM. Statistics: Mann-Whitney U test or One-way ANOVA. (ns: P ≥ 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

Article Snippet: Proteins (40 μg) were separated via SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk in TBST (0.05% Tween-20) for one hour at RT, and incubated overnight at 4 °C with primary antibodies: VEGFR2 (D5B1 Rabbit mAb, 1:1,000, CST #9698), PD-L1 (1:1,000, Abcam #ab213524), and GAPDH (D16H11 XP® Rabbit mAb, 1:1,000, CST #5174).

Techniques: Activation Assay, Disruption, Western Blot, Expressing, In Vitro, Co-Culture Assay, Cell Culture, CCK-8 Assay, Viability Assay, Tube Formation Assay, Irradiation, Staining, MANN-WHITNEY

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 maintains BTIC self-renewal. ( a ) Morphology of self-renewing spheres and immunofluorescence of neuronal and glial lineage markers in differentiated cells. Scale bar=200 μm. ( b ) Immunofluorescence of ING5 in undifferentiated (upper panels) and cells differentiated for 5 days (lower panels). Scale bar=20 μm. ( c , d ) The mRNA levels ( c ) and protein levels ( d ) of ING5 decrease during differentiation in BT 189 cells. n =4. ( e ) Immunoblotting of ING5 protein in the BT 12, and BT 134 lines during differentiation. ( f ) ING5 overexpression increases sphere formation rates and average volumes in BTIC sphere formation assays ( n =3, ** P <0.01, * P <0.05). Scale bar=400 μm. ( g ) ING5 knockdown by siRNA decreases sphere formation rates and sphere sizes of BTICs ( n =4, ** P <0.01, * P <0.05). Scale bars=400 μm. ( h ) (Left) Sphere formation rates for cell lines stably expressing shRNAs against ING5 (shR1 and shR2-ING5) or control non-targeting shRNA (shR-ctr). ( n =4, ** P <0.01, * P <0.05). (Right) Fluorescence of the RFP reporter in stable cell lines superimposed with differential interference contrast (DIC) images. Scale bar=100 μm. ( i ) RT-qPCR analysis of stem cell core transcription factors and stem cell markers after ING5 overexpression. ( j , k ) Western blot analysis of the neural stem cell marker Nestin and neuronal lineage marker Tubb3 in response to ING5 overexpression ( j ) and knockdown ( k ).

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Western Blot, Over Expression, Knockdown, Stable Transfection, Expressing, Control, shRNA, Fluorescence, Quantitative RT-PCR, Marker

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 increases the stem cell pool and inhibits differentiation. ( a ) Flow cytometry analysis of CD133/CD44 positive cells in iPB cell lines (top panels) and CD44 positive cells in shRNA cell lines (bottom panels), gated by isotype control. ( b ) (Left) Mitotic pair analysis of the three division modes: symmetric proliferating (sym-pro), symmetric differentiating (sym-diff) and asymmetric (asym) cell division, in iPB cell lines. Over 150 pairs were counted for each group in one experiment. n =3, * P <0.05. (Right) An example of cell division symmetry based on the distribution of stem cell factor Nestin with the Red arrow indicating symmetric differentiating division and the white arrow asymmetric division. ( c ) Morphological changes of iPB cells before (Day 0) and after (Days 1–3) differentiation induced by 1% FBS. Scale bar=100 μm. ( d ) After differentiation for 5 days, immunofluorescence of Nestin and Tubb3 in shRNA cell lines with an RFP reporter. Scale bar=200 μm. ( e ) Western blot of Nestin and Tubb3 in differentiated shRNA cell lines corresponding to cells shown in d .

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Flow Cytometry, shRNA, Control, Immunofluorescence, Western Blot

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 activates mitogenic pathways to promote self-renewal. ( a ) The sphere formation rates of iPB-ctr and iPB-ING5 overexpressing cells at three successive passages in the absence of EGF and FGF treatment. ( n =3, ** P <0.01, *** P <0.001). ( b ) DIC images of spheres from the tertiary sphere passage in iPB cells. Scale bar=400 μm. ( c ) Sphere formation rate under treatment with protein kinase inhibitors ( n =3, * P <0.05). ( d ) Protein and phosphorylated protein levels of effectors in the PI3K and MEK pathways. Cells were treated with PX-866 at 1 μ M and PD184352 at 2 μ M for 48 h. ( e ) Immunofluorescence of Nestin and Tubb3 in differentiated iPB control (Left panels) or iPB-ING5 cells (Right panels) treated with 1 μ M PX-866 or 2 μ M PD184352. Scale bar=200 μm. ( f ) Flow cytometry analysis of the CD133 positive population in PX-866 (1 μ M ) and PD184352 (2 μ M ) treated BTIC 189 cells, gated by isotype control.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Control, Flow Cytometry

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: The FSH pathway transduces effects of ING5 on stem cell properties. ( a ) Sphere formation assay for cells treated with calcium modulators and FSHR blocking antibody (Anti-FSHR) at the indicated concentrations ( n =3, * P <0.05 and ** P <0.01 compared to iPB-ctr/DMSO; # P <0.05 and ## P <0.01 compared to iPB-ING5/DMSO). ( b ) IPA downstream function analysis indicates the FSH pathway is elevated by ING5. Genes positively correlated with this function were listed with fold changes. ( c ) RT-qPCR of genes related to hormone and steroidogenesis functions. ( n =3, * P <0.05, ** P <0.01) ( d ) The expression levels of FSHB and FSHR genes in BT 189 cells before and after differentiation for 1–5 days. ( e ) Immunostaining for ING5 and FSHR in iPB cells. Scale bar=100 μm. ( f ) Flow cytometry analysis of CD133 positive cells in BT 189 cells treated with FSHR neutralizing antibody or IgG control, gated by isotype control. ( g ) Immunofluorescence of Nestin and Tubb3 shows inhibition of the FSH pathway induces neuronal differentiation. Scale bar=200 μm. ( h ) FSH recombinant protein treatment at indicated concentrations increases sphere formation rates in shRNA cell lines ( n =3, * P <0.05 and ** P <0.01 compared to untreated shR-ctr; # P <0.05 compared to untreated shR-ING cells). ( i ) FSH recombinant protein treatment induces sphere-forming abilities in iPB-ctr cells but not in ING5 overexpressing cells ( n =3, ** P <0.01). ( j ) Sphere formation rates for cells treated with Anti-FSHR or BAPTA alone, and the combination of both ( n =3, * P <0.05). ( k ) FSH treatment at 5 ng/ml for 3 days induces the expression of OCT4 and Nestin in BT 189 cells.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Tube Formation Assay, Blocking Assay, Quantitative RT-PCR, Expressing, Immunostaining, Flow Cytometry, Control, Immunofluorescence, Inhibition, Recombinant, shRNA

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: PHD motif is required for the function of ING5 in BTICs and ING5 levels negatively correlate with survival of GBM. ( a ) Sphere formation assays in iPB cell lines overexpressing wild-type (ING5-FLAG) and PHD-deleted ING5 (ΔPHD) ( n =3, ** P <0.01). ( b ) Western blot shows the protein levels of endogeneous ING5, wildtype ING5 with a FLAG tag and PHD-deleted ING5 (black arrows) in three iPB cell lines. ( c ) ChIP analysis of ING5 binding to promoters of target genes presented as fold enrichment relative to IgG controls. The endogenous ING5 in BT 189 cells, overexpressed ING5 with a Flag tag in iPB-ING5 cells and overexpressed PHD-deleted ING5 protein with a Flag tag were immunoprecipitated by the ING5 antibody and Flag antibody respectively. The upper panels are the schematic representation of the location of the primer sets and promoter regions enriched for ING5 binding were shown in red. ( d ) Kaplan–Meier survival analysis of TCGA GBM patients with high and low levels of ING5 expression (stratified by mean value, n =114). ( e , f ) ING5 expression levels negatively correlate with survival of the Proneural subtype ( n =24) and the Classical subtype ( n =30) of GBM patients. ( g ) The relationship of ING5 levels to survival in the SOX2-low group of patients (ING5, SOX2 stratified by median values, n =61). ( h ) Model for how ING5 functions in the maintenance of BTIC self-renewal. In the absence of growth factors, ING5 induces FSH and calcium signaling by promoting transcription of the FSH receptor and ligand genes, and various plasma membrane calcium channel genes. The FSH and calcium signaling pathways further activate PI3K/AKT and MEK/ERK signaling to induce stem cell features and the expression of stemness factors OCT4, OLIG2 and Nestin. Gene activation by ING5 is dependent on its PHD motif to target ING5-associated histone acetyltransferase complexes to the promoters.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Western Blot, FLAG-tag, Binding Assay, Immunoprecipitation, Expressing, Clinical Proteomics, Membrane, Protein-Protein interactions, Activation Assay

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 1. Upregulation of ALDH1A3 in oxGBM cells is associated with the increased expression and release of pro-angiogenic factors PAI-1 and IL-8. GBM cell lines were transduced with ALDH1A3 for overexpression (oxGBM) or with empty vector (evGBM). (A) Confirmation of up-regulation of ALDH1A3 mRNA level in oxGBM cells by RT2-PCR. (B) Confirmation of up-regulation of ALDH1A3 protein expression by Western blot. wt, wild-type cells. The uncropped blots are shown in Figure S7; (C) Angiogenesis array. The blots showed duplicated dots for 55 angiogenesis-related proteins in the media of oxU373 or evU373. 10 of 55 proteins were upregulated more than 2-fold in ox group compared to ev group (indicated by rectangle). They are: (1) Ang-1, (2) artemin, (3) TF, (4) ET-1, (5) GM-CSF, (6) IL-8, (7) PDGF-AA, (8) PAI-1, (9) PEDF, and (10) uPA. (D) Semi-quantification of the dots representing PAI-1 and IL-8. (E) Immunofluorescence staining of GBM cells. U373 (left panel) and LN229 (right panel). Co-localization of ALDH1A3 with PAI-1 and IL-8 was observed in oxGBM cells, whereas no immunoreactivity of PAI-1 and IL-8 was detected in evGBM cells. (F) mRNA expression of PAI-1 and IL-8 in transduced U373 cells and the effect of inhibitors. Tiplaxtinin (Tip, 30 µM) and reparixin (Rep, 1 µM) are the specific inhibitors of PAI-1 and IL-8 receptors CXCR1/2, respectively. (G) Detection of PAI-1 and potential signaling proteins by Western blot. IOD: optical density. The uncropped blots are shown in Figure S8. **, p < 0.01; ***, p < 0.001, compared with ev. ##, p < 0.01; ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 2. Overexpression of ALDH1A3 in GBM cells activated endothelial angiogenesis in indirect co-culture with endothelial cells (ECs), which was reversed by treatment with respective inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Indirect co-culture was performed by culture of HBMECs in a conditioned medium (CM) containing the media derived from evGBMs or oxGBMs and ECGM in a ratio of 1:1. PAI-1 inhibitor tiplaxtinin (Tip, 30 µM) and CXCR1/2 inhibitor reparixin (Rep, 1 µM) or vehicle DMSO (0.1%) was added to CM, followed by EC behavior study. All data were reproduced in three independent experiments. (A) Proliferation assay in HBMEC and HUVEC. Indirect co-culture of HBMECs and HUVECs with CM derived from oxU373 and oxLN229 stimulated EC proliferation, which was completely reversed by the treatment of tiplaxtinin, not by reparixin. (B) Scratch assay in HBMEC. Left panel: images were acquired 24 h after scratching. Scale bar: 200 µm. Right panel: quantitative analysis. Culture of HBMECs with CM derived from oxU373 or oxLN229 (oxCM) significantly promoted HBMEC migration, which was reversed by the treatment of tiplaxtinin and reparixin, respectively. (C) Transwell invasion assay in HBMEC. Left panel: Representative images of invaded cells were acquired after 24 h of incubation. Scale bar: 100 µm. Right panel: quantitative analysis. Culture of HBMECs with oxCM accelerated HBMEC invasion. This effect was significantly inhibited by the treatment of reparixin but not by tiplaxtinin. (D) Tube formation assay in HBMEC. Left panel: representative images of tube formation. Scale bar: 200 µm. Right panel: quantitative analysis of branching points per field. Tube formation in HBMECs was stimulated by incubation with oxCM, which was completely diminished by both inhibitors. (E) Sprouting assay in HBMEC. Left panel: representative images of sprouting in HBMECs after 24 h of co-culture. Scale bar: 100 µm. A pronounced increase in sprouting was observed in HBMECs cultured in oxCM. Tiplaxtinin and reparixin suppressed the sprouting effect resulting from oxCM. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with evCM. #, p < 0.05; ##, p < 0.01 and ###, p < 0.001, compared with oxCM.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Over Expression, Co-Culture Assay, Derivative Assay, Proliferation Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Incubation, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 4. oxGBM-derived culture media stimulated angiogenesis on CAM, which was fully rescued by the treatment of tiplaxtinin and reparixin. CAM was incubated with the culture media derived from evU373 or oxU373 cells with or without tiplaxtinin (Tip, 30 µM) or reparixin (Rep, 1 µM) or DMSO (as a vehicle control, 0.1%) for 72 h. (A) Microscopy view of the vasculature structure on CAM. More enriched microvessel network was clearly visible in the ox group, which was significantly reduced in tiplaxtinin- and reparixin-treated CAMs. The images were acquired using a stereo microscope on ED13 (scale bar: 1 mm). Stem vessel (arrow); branched microvessel network (arrow heads). (B) Histological features of CAM after hematoxylin-eosin (H&E) staining. The CAM consists of the chorionic epithelium layer (ChE), allantoic epithelium (AE) layer, and the mesenchymal (MES) layer (arrows). Microvessel (arrowheads) density was much higher in the MES layer of ox section compared to ev section, which was clearly reduced in tiplaxtinin- and reparixin-treated sections. Scale bar: 50 µm. (C) Quantitative analysis of branching point of vessels based on microscopy images. The number of branching points and microvessels was counted by the ImageJ software (v1.1.53t) in 3 fields/CAM (n = 10 CAM/group). (D) Quantitative analysis of microvessel numbers based on H&E-stained CAM sections. Microvessel number was counted manually on H&E-stained CAMs. 10 fields/section (n = 6 sections/group). ***, p < 0.001, compared with ev. ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Incubation, Control, Microscopy, Staining, Software

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: Endothelial cell tubule formation assay. BME cells (3 × 105/ml) were seeded onto type I collagen gels in medium supplemented with 10% calf serum. On the following day, the medium was aspirated and replaced with conditioned serum-free medium derived from cocultures of G8 myoblast monolayers and TA3wt (A) TA3sCD44 (B), TA3sCD44/MMP-9-CD44fp (C), or TA3sCD44/MMP-9v5 (D) cells. A total of 30 μg/ml of pan specific anti-TGF-β (E) or anti-bFGF (F) antibody were added to the TA3wt/G8-conditioned coculture medium prior to use in the assay. Tubules are indicated by arrows. Bar, 140 μm.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Tube Formation Assay, Derivative Assay

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: CD44-anchored MMP-9 activates TGF-β. (A) The ability of TA3 transfectant-G8 myoblast coculture media to stimulate TMLC luciferase activity is shown. Conditioned coculture media tested are indicated. (B) Pan-specific TGF-β antibody (30 μg/ml) or specific antibodies against TGF-β1 (100 ng/ml), TGF-β2(100ng/ml), or TGF-β3(100 ng/ml) were used to determine the activity of TGF-β isoforms in the coculture media. One unit of luciferase activity corresponds to the activity produced by 5 pg of purified human TGF-β1 (R & D).

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Produced, Purification

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: TGF-β is activated by purified MMP-9. (A) TMLC luciferase activity induced by concentrated COS cell supernatants containing the indicated latent TGF-β isoforms following incubation with the indicated purified AMPA-activated MMPs. Comparable amounts of latent TGF-β1, TGF-β2, and TGF-β3 were present in the supernatants as assessed by both Western blot analysis and TMLC-luciferase induction following heat treatment (80°C for 5min) (B) TMLC luciferase activity induced by affinity-purified TGF-β2 following incubation with the indicated purified activated MMPs. Activity is expressed in relative light units (RLU), in which 800 RLU corresponds to the luciferase activity generated by 1 pg of purified human TGF-β1 (R & D). (C) TGF-β is proteolytically cleaved by MMP-9 and MMP-2. Purified v5-tagged TGF-β2 (lane 1) was incubated with protein-A Sepharose-bound, AMPA-activated MMP-9 (lane 2), MMP-2 (lane 3), and MMP-3 (lane 4) for 90 min at 37°C. Following incubation, the supernatants were separated from the beads, subjected to SDS/12% PAGE, transferred to Hybond-C membranes, and blotted with anti-v5 antibody. (D) MMP-2/CD44 fusion protein expression promotes TGF-β activation in TA3sCD44 cells. TMLC luciferase assays were performed with serum-free coculture media from TA3sCD44 cells transiently transfected with the indicated cDNAs.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Purification, Luciferase, Activity Assay, Incubation, Western Blot, Affinity Purification, Generated, Expressing, Activation Assay, Transfection

Journal: Heliyon

Article Title: Qizhu Anti-Cancer Recipe promotes anoikis of hepatocellular carcinoma cells by activating the c-Jun N-terminal kinase pathway

doi: 10.1016/j.heliyon.2023.e22089

Figure Lengend Snippet: The effects of Qizhu Anti-Cancer Recipe on apoptotic proteins and angiopoiesis in hepatocellular carcinoma cells. (A–G) The effects of 3.72 μg/ml sorafenib or QACR (0 μg/ml; 20 μg/ml; 40 μg/ml; 200 μg/ml) on the expression of proliferating cell nuclear antigen (PCNA), Bcl-2, caspase-3, caspase-8, caspase-9, and CD31 according to Western blot analysis. (H) Angiogenesis in MHCC97-L or SK-Hep-1 cells and HUVECs according to tube formation assay. Data are presented as the mean ± SD of triplicate experiments, one-way ANOVA and two-way ANOVA were used for statistical test. Compared to the NC group,*P < 0.05, **P ＜ 0.01, ***P ＜ 0.001.

Article Snippet: The tissues were dewaxed and rehydrated using graded ethanol, followed by incubation at 4 °C overnight with primary antibodies against mouse anti-PCNA (1:10000; Abcam), rabbit anti-Bcl-2 (1:100; Abcam), rabbit anti-Caspase-3 (1:100; Abcam), rabbit anti-Caspase-8 (1:250; Abcam), rabbit anti-Caspase-9 (1:300; Abcam), rabbit anti-CD31 (1:250; Abcam), and rabbit anti- p -JNK (1:50; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Tube Formation Assay

Journal: Heliyon

Article Title: Qizhu Anti-Cancer Recipe promotes anoikis of hepatocellular carcinoma cells by activating the c-Jun N-terminal kinase pathway

doi: 10.1016/j.heliyon.2023.e22089

Figure Lengend Snippet: The effects of Qizhu Anti-Cancer Recipe on tumor growth and angiopoiesis in vivo (A) The effects of QACR (0 g/kg; 10.4 g/kg; 20.8 g/kg; 41.6 g/kg) in vivo tumor growth according to Bioluminescence imaging. (B) Representative image of liver tumor obtained from mice in each group. (C) The expression of PCNA, Bcl-2, CD31, caspase-3, caspase-8, and caspase-9 in each group according to immunohistochemical analysis. (D) The positive rate of PCNA, Bcl-2, CD31, caspase-3, caspase-8, CD31, and caspase-9 in each group. Data are presented as the mean ± SD of five independent experiments, one-way ANOVA was used for statistical test. Compared to the control group,*P < 0.05, **P ＜ 0.01, ***P ＜ 0.001.

Article Snippet: The tissues were dewaxed and rehydrated using graded ethanol, followed by incubation at 4 °C overnight with primary antibodies against mouse anti-PCNA (1:10000; Abcam), rabbit anti-Bcl-2 (1:100; Abcam), rabbit anti-Caspase-3 (1:100; Abcam), rabbit anti-Caspase-8 (1:250; Abcam), rabbit anti-Caspase-9 (1:300; Abcam), rabbit anti-CD31 (1:250; Abcam), and rabbit anti- p -JNK (1:50; Cell Signaling Technology).

Techniques: In Vivo, Imaging, Expressing, Immunohistochemical staining, Control

Journal: Heliyon

Article Title: Qizhu Anti-Cancer Recipe promotes anoikis of hepatocellular carcinoma cells by activating the c-Jun N-terminal kinase pathway

doi: 10.1016/j.heliyon.2023.e22089

Figure Lengend Snippet: The effects of Qizhu Anti-Cancer Recipe on proliferation, apoptosis, angiopoiesis -related proteins in vivo (A) The expression of PCNA, Bcl-2, CD31, caspase-3, caspase-8, and caspase-9 in tumor tissues obtained from each group according to Western blot. (B–D) The densitometry analysis of PCNA, Bcl-2, CD31, caspase-3, caspase-8, and caspase-9. Data are presented as the mean ± SD of five independent experiments, one-way ANOVA was used for statistical test. Compared to the control group,*P < 0.05, **P ＜ 0.01, ***P ＜ 0.001.

Article Snippet: The tissues were dewaxed and rehydrated using graded ethanol, followed by incubation at 4 °C overnight with primary antibodies against mouse anti-PCNA (1:10000; Abcam), rabbit anti-Bcl-2 (1:100; Abcam), rabbit anti-Caspase-3 (1:100; Abcam), rabbit anti-Caspase-8 (1:250; Abcam), rabbit anti-Caspase-9 (1:300; Abcam), rabbit anti-CD31 (1:250; Abcam), and rabbit anti- p -JNK (1:50; Cell Signaling Technology).

Techniques: In Vivo, Expressing, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Knockdown of EGFR inhibits angiogenesis in Cr(VI)-transformed cells. A, expression of p-EGFR was examined using immunoblotting to confirm stable knockdown of EGFR (shEGFR) in Cr(VI)-transformed cells (BEAS-2B-Cr). B, representative images of tube formation in HUVECs (left panel) and quantitation of tube branches (right panel) induced by conditioned medium from BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown. *, p < 0.05, compared with scramble cells. C, chick embryos were exposed to conditioned medium from BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown for 72 h. Top panel, CAM tissue directly beneath each Matrigel graft was photographed. The image represents at least three chick embryos. Bottom panel, quantitation of vascular branches in CAM. The results were expressed as means ± S.D. *, p < 0.05, compared with scramble cells. D, in vivo Matrigel plug assay. Top panel, representative pictures of Matrigel plugs from BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown at day 14 after inoculation into mice. Bottom panel, hemoglobin level of Matrigel plugs. The data are means ± S.D. from replicate experiments (n = 3). *, p < 0.05, compared with scramble cells.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Transformation Assay, Expressing, Western Blot, Quantitation Assay, In Vivo, Matrigel Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Knockdown of EGFR reduces MMP-1 and VEGF expression in Cr(VI)-transformed cells. A, Cr(VI)-transformed cells (BEAS-2B-Cr) with (shEGFR) or without (Scramble) stable knockdown of EGFR were examined for MMP-1 and VEGF expression using immunoblotting analysis. B, VEGF level was measured in BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown using ELISA. *, p < 0.05, compared with scramble cells. C, Cr(VI)-transformed cells with (shEGFR) or without (Scramble) stable EGFR knockdown were grown in chamber slides. Expression of MMP-1 (green) and VEGF (red) was examined using immunofluorescence staining. The images were captured using fluorescence microscope. Expression of DAPI (blue) was used as nuclear control. D, quantitative PCR analysis. mRNA levels of angiogenin, IL-6, MMP-1, and GM-CSF were examined in BEAS-2B-Cr cells with (shEGFR) or without (Scramble) stable EGFR knockdown. *, p < 0.05, compared with scramble cells.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Expressing, Transformation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Microscopy, Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Inhibition of EGFR down-regulates HIF-1α and p38 in Cr(VI)-transformed cells. A, Cr(VI)-transformed cells (BEAS-2B-Cr) and passage-matched normal ones (BEAS-2B) were collected for immunoblotting analysis. B, immunofluorescence staining analysis in normal lung tissue from a health patient and in lung tumor tissue and its normal adjacency from a worker exposed to Cr(VI) for 19 years as described under “Experimental Procedures.” C, binding of HIF-1α to hypoxia response element region of VEGF promoter was examined using ChIP assay. Data are means ± S.D. (n = 3). * and #, p < 0.05, compared with BEAS-2B cells and BEAS-2B-Cr scramble cells. D, BEAS-2B-Cr cells with (shEGFR) and without (Scramble) (left panel) and tumor tissues injected with stable knockdown EGFR (shEGFR) and scramble (right panel) were subjected to immunoblotting analysis. E, BEAS-2B-Cr cells were treated with SB203580 or PX478 for 24 h. Whole cell lysates were harvested for immunoblotting analysis. F, tube formation assay. Conditioned medium were collected from BEAS-2B-Cr cells treated with SB203580 or PX478 for 24 h or without treatment. HUVECs were plated onto Matrigel-coated plates and incubated with those conditioned medium. Top panel, representative images from each treatment group. Bottom panel, quantitative results of tube formation are shown as mean ± S.D. (n = 3). *, p < 0.05, compared with BEAS-2B-Cr cells without treatment. G, BEAS-2B-Cr cells were transient transfected with HIF-1α shRNA for 48 h. Whole cell lysates were harvested for immunoblotting analysis. H, Cr(VI)-transformed cells were transfected with EGFR expression plasmid and/or EGFR shRNA for 48 h in various combinations. The cells were collected, and whole cell lysates were subjected for immunoblotting analysis.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Transformation Assay, Western Blot, Immunofluorescence, Staining, Binding Assay, Injection, Knockdown, Tube Formation Assay, Incubation, Transfection, shRNA, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Schematic mechanism of angiogenesis and tumorigenesis of Cr(VI)-transformed cells. Our previous study has demonstrated that chronic exposure of cells to Cr(VI) causes malignant cell transformation and EGFR is constitutively activated in Cr(VI)-transformed cells. The present study has found that constitutive activation of EGFR causes activations of p38 and HIF-1α, leading to angiogenesis and tumorigenesis of Cr(VI)-transformed cells.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Transformation Assay, Activation Assay