g7 system Search Results


recombinant mouse gdf7 protein  (R&D Systems)
92
R&D Systems recombinant mouse gdf7 protein
<t>GDF7</t> alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with <t>rmGDF7</t> (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.
Recombinant Mouse Gdf7 Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/Recombinant+Mouse+GDF-7+Protein/pmc09800101-29-0-23
Average 92 stars, based on 1 article reviews
recombinant mouse gdf7 protein - by Bioz Stars, 2026-09
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roc atcc g 7  (ATCC)
94
ATCC roc atcc g 7
<t>GDF7</t> alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with <t>rmGDF7</t> (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.
Roc Atcc G 7, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/G-7/le_coupanec_alain__2018__consequences_neurologiques_des_clivages_de_la_glycoproteine_s_du_coronavirus_neuroinvasif_humain_oc43-63-45-45
Average 94 stars, based on 1 article reviews
roc atcc g 7 - by Bioz Stars, 2026-09
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e g7 ova cell lines  (ATCC)
96
ATCC e g7 ova cell lines
<t>GDF7</t> alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with <t>rmGDF7</t> (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.
E G7 Ova Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/E%2EG7-OVA/pmc13109015-654-4-10
Average 96 stars, based on 1 article reviews
e g7 ova cell lines - by Bioz Stars, 2026-09
96/100 stars
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g7 3 97 ok235615  (ATCC)
95
ATCC g7 3 97 ok235615
<t>GDF7</t> alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with <t>rmGDF7</t> (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.
G7 3 97 Ok235615, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/G-7/pm37110263-162-143-156
Average 95 stars, based on 1 article reviews
g7 3 97 ok235615 - by Bioz Stars, 2026-09
95/100 stars
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continuous wave laser  (Coherent Corp)
96
Coherent Corp continuous wave laser
<t>GDF7</t> alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with <t>rmGDF7</t> (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.
Continuous Wave Laser, supplied by Coherent Corp, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/Verdi/10__5194_slash_amt___14___7693___2021-55-7-11
Average 96 stars, based on 1 article reviews
continuous wave laser - by Bioz Stars, 2026-09
96/100 stars
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anti von hippel lindau vhl  (Proteintech)
93
Proteintech anti von hippel lindau vhl
Expression of von <t>Hippel</t> <t>Lindau</t> protein in von Hippel Lindau overexpression and von Hippel Lindau knockdown cell models under normoxia condition. A: Western blot assay was performed to detect von Hippel Lindau <t>(VHL)</t> protein expression in cell models of control group, overexpressed empty carrier (OE-NC) group and VHL overexpressed (OE-VHL) group under normoxia condition; B: Statistical analysis of VHL protein expression in cell models of control group, OE-NC group and OE-VHL group under normoxia condition; C: Western blot assay was performed to detect VHL protein expression in cell models of control group, knockdown empty vector (sh-NC) group and VHL knockdown (sh-VHL) group under normoxia condition; D: Statistical analysis of VHL protein expression in cell models of control group, sh-NC group and sh-VHL group under normoxia condition. a P < 0.05; b P < 0.01. VHL: von Hippel Lindau; OE-NC: Overexpressed empty carrier; OE-VHL: VHL overexpressed; sh-NC: Knockdown empty vector; sh-VHL: VHL knockdown.
Anti Von Hippel Lindau Vhl, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/VHL+Antibody/pmc11185293-106-213-218
Average 93 stars, based on 1 article reviews
anti von hippel lindau vhl - by Bioz Stars, 2026-09
93/100 stars
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el4 expressing chicken ovalbumin  (ATCC)
94
ATCC el4 expressing chicken ovalbumin
Figure 2 TCR-mediated virus-cell entry is highly specific and requires high-affinity interaction between the TCR and its cognate MHC/peptide complex. (a) Infection of <t>EL4</t> cells by MV-eGFP, MV-T7, MV-m33, and MV-CD38 in the absence or presence of 5 106 M pOV8 (SIINFEKL) or SIYR peptide. Only the MV displaying the high-affinity m33 TCR infected these H-2b cells presenting the SIYR peptide. None of the viruses infected cells presenting the null peptide pOV8. Scale bar ¼ 300 mm. (b) Percentage of GFP positive after infection of SIYR or pOV8-loaded (3 106 M) EL4 (H-2b) and P815 (H-2d) cells with MV-m33. Infection of SIYR/EL4 cells by MV-m33 was efficiently inhibited by addition of anti-Kb antibody (50 mg/ml). Bars represent duplicate experiment.
El4 Expressing Chicken Ovalbumin, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/E%2EG7-OVA%3B+Lymphoma%3B+Mouse/pm15215883-70-5-10
Average 94 stars, based on 1 article reviews
el4 expressing chicken ovalbumin - by Bioz Stars, 2026-09
94/100 stars
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bmp12 gdf7  (R&D Systems)
93
R&D Systems bmp12 gdf7
Figure 2 TCR-mediated virus-cell entry is highly specific and requires high-affinity interaction between the TCR and its cognate MHC/peptide complex. (a) Infection of <t>EL4</t> cells by MV-eGFP, MV-T7, MV-m33, and MV-CD38 in the absence or presence of 5 106 M pOV8 (SIINFEKL) or SIYR peptide. Only the MV displaying the high-affinity m33 TCR infected these H-2b cells presenting the SIYR peptide. None of the viruses infected cells presenting the null peptide pOV8. Scale bar ¼ 300 mm. (b) Percentage of GFP positive after infection of SIYR or pOV8-loaded (3 106 M) EL4 (H-2b) and P815 (H-2d) cells with MV-m33. Infection of SIYR/EL4 cells by MV-m33 was efficiently inhibited by addition of anti-Kb antibody (50 mg/ml). Bars represent duplicate experiment.
Bmp12 Gdf7, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/Recombinant+Human+GDF-7+Protein%2C+CF/pm37381820-262-37-38
Average 93 stars, based on 1 article reviews
bmp12 gdf7 - by Bioz Stars, 2026-09
93/100 stars
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novus biologicals nbp128031  (Novus Biologicals)
94
Novus Biologicals novus biologicals nbp128031
Figure 2 TCR-mediated virus-cell entry is highly specific and requires high-affinity interaction between the TCR and its cognate MHC/peptide complex. (a) Infection of <t>EL4</t> cells by MV-eGFP, MV-T7, MV-m33, and MV-CD38 in the absence or presence of 5 106 M pOV8 (SIINFEKL) or SIYR peptide. Only the MV displaying the high-affinity m33 TCR infected these H-2b cells presenting the SIYR peptide. None of the viruses infected cells presenting the null peptide pOV8. Scale bar ¼ 300 mm. (b) Percentage of GFP positive after infection of SIYR or pOV8-loaded (3 106 M) EL4 (H-2b) and P815 (H-2d) cells with MV-m33. Infection of SIYR/EL4 cells by MV-m33 was efficiently inhibited by addition of anti-Kb antibody (50 mg/ml). Bars represent duplicate experiment.
Novus Biologicals Nbp128031, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/CD90%2FThy1+Antibody+(G7)+-+Azide+and+BSA+Free/pm41858055-213-35-35
Average 94 stars, based on 1 article reviews
novus biologicals nbp128031 - by Bioz Stars, 2026-09
94/100 stars
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mouse monoclonal anti flag  (Novus Biologicals)
91
Novus Biologicals mouse monoclonal anti flag
Figure 2 TCR-mediated virus-cell entry is highly specific and requires high-affinity interaction between the TCR and its cognate MHC/peptide complex. (a) Infection of <t>EL4</t> cells by MV-eGFP, MV-T7, MV-m33, and MV-CD38 in the absence or presence of 5 106 M pOV8 (SIINFEKL) or SIYR peptide. Only the MV displaying the high-affinity m33 TCR infected these H-2b cells presenting the SIYR peptide. None of the viruses infected cells presenting the null peptide pOV8. Scale bar ¼ 300 mm. (b) Percentage of GFP positive after infection of SIYR or pOV8-loaded (3 106 M) EL4 (H-2b) and P815 (H-2d) cells with MV-m33. Infection of SIYR/EL4 cells by MV-m33 was efficiently inhibited by addition of anti-Kb antibody (50 mg/ml). Bars represent duplicate experiment.
Mouse Monoclonal Anti Flag, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/DYKDDDDK+Epitope+Tag+Antibody+(29E4%2EG7)/pmc10601634-54-69-72
Average 91 stars, based on 1 article reviews
mouse monoclonal anti flag - by Bioz Stars, 2026-09
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vhl  (MedChemExpress)
92
MedChemExpress vhl
Figure 2 TCR-mediated virus-cell entry is highly specific and requires high-affinity interaction between the TCR and its cognate MHC/peptide complex. (a) Infection of <t>EL4</t> cells by MV-eGFP, MV-T7, MV-m33, and MV-CD38 in the absence or presence of 5 106 M pOV8 (SIINFEKL) or SIYR peptide. Only the MV displaying the high-affinity m33 TCR infected these H-2b cells presenting the SIYR peptide. None of the viruses infected cells presenting the null peptide pOV8. Scale bar ¼ 300 mm. (b) Percentage of GFP positive after infection of SIYR or pOV8-loaded (3 106 M) EL4 (H-2b) and P815 (H-2d) cells with MV-m33. Infection of SIYR/EL4 cells by MV-m33 was efficiently inhibited by addition of anti-Kb antibody (50 mg/ml). Bars represent duplicate experiment.
Vhl, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/VHL%2C+Human/pmc11350581__au4c00278_si_001-2-8-14
Average 92 stars, based on 1 article reviews
vhl - by Bioz Stars, 2026-09
92/100 stars
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gdf7  (R&D Systems)
93
R&D Systems gdf7
FIGURE 2. Characterization of recombinant GDF6, <t>GDF7,</t> and BMP10. A, immuno- blotting analyses of recombinant GDF6 and GDF7. Recombinant proteins were analyzed under reduced or nonreduced conditions following blotting with specific antibodies. B, biochemical properties of purified recombinant human BMP10. BMP10 with an N-termi- nal His6 tag was purified from conditioned media of 293T cells using metal chelate chro- matography. BMP10 was detected by Coomassie Blue staining (lanes 1–3) and immuno- blotting using antibodies against polyhistidine His6 (lanes 4 and 5) under nonreduced or reduced conditions. Some samples were treated with peptide N-glycosidase F (PNGase) to remove N-linked carbohydrate side chains.
Gdf7, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/g7+system/Recombinant+Human+GDF-7+Protein%2C+CF/10__1074_slash_jbc__m504629200-56-2-14
Average 93 stars, based on 1 article reviews
gdf7 - by Bioz Stars, 2026-09
93/100 stars
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Image Search Results


GDF7 alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: Injection, Saline, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay

GDF7 inhibits inflammation and oxidative stress in LPS-treated mice. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, BALF was collected for the analysis of total cells, neutrophils, and macrophages ( n = 6). (b) MPO activity in lung tissues ( n = 6). (c) IL-6 and TNF- α levels in lung tissues ( n = 6). (d) ROS content in lung tissues ( n = 6). (e) MDA and 4-HNE levels in lung tissues ( n = 6). (f-g) Total SOD activity and GSH content in lung tissues ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 inhibits inflammation and oxidative stress in LPS-treated mice. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, BALF was collected for the analysis of total cells, neutrophils, and macrophages ( n = 6). (b) MPO activity in lung tissues ( n = 6). (c) IL-6 and TNF- α levels in lung tissues ( n = 6). (d) ROS content in lung tissues ( n = 6). (e) MDA and 4-HNE levels in lung tissues ( n = 6). (f-g) Total SOD activity and GSH content in lung tissues ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: Injection, Saline, Activity Assay

GDF7 reduces LPS-stimulated inflammation and oxidative stress in primary macrophages. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (b) ROS content in macrophages ( n = 6). (c) MDA and 4-HNE levels in macrophages ( n = 6). (d-e) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 reduces LPS-stimulated inflammation and oxidative stress in primary macrophages. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (b) ROS content in macrophages ( n = 6). (c) MDA and 4-HNE levels in macrophages ( n = 6). (d-e) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: Activity Assay

GDF7 attenuates LPS-induced ALI through activating AMPK in vivo. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, lung tissues were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (b) To inhibit AMPK, ALI mice were intraperitoneally injected with CpC (20 mg/kg) at 2 h pre- and 2 h post-rmGDF7 injection, and then IL-6 and TNF- α levels in lung tissues were detected ( n = 6). (c) ROS content in lung tissues ( n = 6). (d) MDA and 4-HNE levels in lung tissues ( n = 6). (e) LDH activity in lung tissues ( n = 6). (f) Lung wet to dry ratio ( n = 6). (g) Arterial blood gas analysis results ( n = 6). (h) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 attenuates LPS-induced ALI through activating AMPK in vivo. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, lung tissues were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (b) To inhibit AMPK, ALI mice were intraperitoneally injected with CpC (20 mg/kg) at 2 h pre- and 2 h post-rmGDF7 injection, and then IL-6 and TNF- α levels in lung tissues were detected ( n = 6). (c) ROS content in lung tissues ( n = 6). (d) MDA and 4-HNE levels in lung tissues ( n = 6). (e) LDH activity in lung tissues ( n = 6). (f) Lung wet to dry ratio ( n = 6). (g) Arterial blood gas analysis results ( n = 6). (h) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vivo, Injection, Saline, Western Blot, Activity Assay

GDF7 attenuates LPS-induced ALI through activating AMPK in vitro. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. To inhibit AMPK, macrophages were pretreated with CpC (10 μ mol/L) for 12 h before LPS stimulation. Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (b) ROS content in macrophages ( n = 6). (c) MDA and 4-HNE levels in macrophages ( n = 6). (d-e) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 attenuates LPS-induced ALI through activating AMPK in vitro. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. To inhibit AMPK, macrophages were pretreated with CpC (10 μ mol/L) for 12 h before LPS stimulation. Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (b) ROS content in macrophages ( n = 6). (c) MDA and 4-HNE levels in macrophages ( n = 6). (d-e) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vitro, Activity Assay

GDF7 activates AMPK through downregulating STING in vivo. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, lung tissues were harvested for the analysis of STING using western blot ( n = 6). (b) To investigate the involvement of STING, STING KO mice were used, and lung tissues were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (c) IL-6 and TNF- α levels in the lung tissues ( n = 6). (d) ROS content in lung tissues ( n = 6). (e) LDH activity in lung tissues ( n = 6). (f) Lung wet to dry ratio ( n = 6). (g) Arterial blood gas analysis results ( n = 6). (h) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant. NS indicated no statistical significance.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 activates AMPK through downregulating STING in vivo. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, lung tissues were harvested for the analysis of STING using western blot ( n = 6). (b) To investigate the involvement of STING, STING KO mice were used, and lung tissues were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (c) IL-6 and TNF- α levels in the lung tissues ( n = 6). (d) ROS content in lung tissues ( n = 6). (e) LDH activity in lung tissues ( n = 6). (f) Lung wet to dry ratio ( n = 6). (g) Arterial blood gas analysis results ( n = 6). (h) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant. NS indicated no statistical significance.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vivo, Injection, Saline, Western Blot, Activity Assay

GDF7 activates AMPK through downregulating STING in vitro. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. To investigate the involvement of STING, STING KO macrophages were used. Cells were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (b) Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (c) ROS content in macrophages ( n = 6). (d) MDA and 4-HNE levels in macrophages ( n = 6). (e-f) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant. NS indicated no statistical significance.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 activates AMPK through downregulating STING in vitro. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. To investigate the involvement of STING, STING KO macrophages were used. Cells were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (b) Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (c) ROS content in macrophages ( n = 6). (d) MDA and 4-HNE levels in macrophages ( n = 6). (e-f) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant. NS indicated no statistical significance.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vitro, Western Blot, Activity Assay

Schematic diagram of the molecular mechanisms underlying GDF7-regulatedsepsis-induced ALI.

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: Schematic diagram of the molecular mechanisms underlying GDF7-regulatedsepsis-induced ALI.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques:

Expression of von Hippel Lindau protein in von Hippel Lindau overexpression and von Hippel Lindau knockdown cell models under normoxia condition. A: Western blot assay was performed to detect von Hippel Lindau (VHL) protein expression in cell models of control group, overexpressed empty carrier (OE-NC) group and VHL overexpressed (OE-VHL) group under normoxia condition; B: Statistical analysis of VHL protein expression in cell models of control group, OE-NC group and OE-VHL group under normoxia condition; C: Western blot assay was performed to detect VHL protein expression in cell models of control group, knockdown empty vector (sh-NC) group and VHL knockdown (sh-VHL) group under normoxia condition; D: Statistical analysis of VHL protein expression in cell models of control group, sh-NC group and sh-VHL group under normoxia condition. a P < 0.05; b P < 0.01. VHL: von Hippel Lindau; OE-NC: Overexpressed empty carrier; OE-VHL: VHL overexpressed; sh-NC: Knockdown empty vector; sh-VHL: VHL knockdown.

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Expression of von Hippel Lindau protein in von Hippel Lindau overexpression and von Hippel Lindau knockdown cell models under normoxia condition. A: Western blot assay was performed to detect von Hippel Lindau (VHL) protein expression in cell models of control group, overexpressed empty carrier (OE-NC) group and VHL overexpressed (OE-VHL) group under normoxia condition; B: Statistical analysis of VHL protein expression in cell models of control group, OE-NC group and OE-VHL group under normoxia condition; C: Western blot assay was performed to detect VHL protein expression in cell models of control group, knockdown empty vector (sh-NC) group and VHL knockdown (sh-VHL) group under normoxia condition; D: Statistical analysis of VHL protein expression in cell models of control group, sh-NC group and sh-VHL group under normoxia condition. a P < 0.05; b P < 0.01. VHL: von Hippel Lindau; OE-NC: Overexpressed empty carrier; OE-VHL: VHL overexpressed; sh-NC: Knockdown empty vector; sh-VHL: VHL knockdown.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Control, Plasmid Preparation

Effects of Thymoquinone on ubiquitination degradation of hypoxia-inducible factor-1α protein mediated by von Hippel Lindau under normoxia condition. A: Western blot analysis was performed to detect the expression of von Hippel Lindau (VHL) protein and hypoxia-inducible factor-1α (HIF-1α) protein in Lenti-VHL group, Lenti-VHL + Thymoquinone (TQ) group, VHL knockdown (sh-VHL) group and sh-VHL+TQ group under normoxia condition; B: Statistical analysis of VHL protein expression in the Lenti-VHL group, Lenti-VHL+TQ group, sh-VHL group and sh-VHL+TQ group under normoxia condition; C: Statistical analysis of HIF-1α expression in Lenti-VHL group, Lenti-VHL+TQ group, sh-VHL group and sh-VHL+TQ group under normoxia condition. a P < 0.05; b P < 0.01. TQ: Thymoquinone; VHL: von Hippel Lindau; sh-VHL: VHL knockdown; HIF-1α: Hypoxia-inducible factor-1α.

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Effects of Thymoquinone on ubiquitination degradation of hypoxia-inducible factor-1α protein mediated by von Hippel Lindau under normoxia condition. A: Western blot analysis was performed to detect the expression of von Hippel Lindau (VHL) protein and hypoxia-inducible factor-1α (HIF-1α) protein in Lenti-VHL group, Lenti-VHL + Thymoquinone (TQ) group, VHL knockdown (sh-VHL) group and sh-VHL+TQ group under normoxia condition; B: Statistical analysis of VHL protein expression in the Lenti-VHL group, Lenti-VHL+TQ group, sh-VHL group and sh-VHL+TQ group under normoxia condition; C: Statistical analysis of HIF-1α expression in Lenti-VHL group, Lenti-VHL+TQ group, sh-VHL group and sh-VHL+TQ group under normoxia condition. a P < 0.05; b P < 0.01. TQ: Thymoquinone; VHL: von Hippel Lindau; sh-VHL: VHL knockdown; HIF-1α: Hypoxia-inducible factor-1α.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Knockdown

Further validation of the effects of Thymoquinone on ubiquitination degradation of hypoxia-inducible factor-1αprotein. A: Western blot assay was performed to detect hypoxia-inducible factor-1α (HIF-1α) expression in von Hippel Lindau (VHL) knockdown PANC-1 cells treated with cycloheximide (CHX) at 0 h, 1 h, 2 h and 3 h under normoxia condition; B: Statistical analysis of HIF-1α protein expression in VHL knockdown PANC-1 cells treated with CHX at 0 h, 1 h, 2 h and 3 h under normoxia condition; C: Western blot assay was performed to detect HIF-1α expression in VHL knockdown PANC-1 cells treated with CHX + Thymoquinone (TQ) at 0 h, 1 h, 2 h and 3 h under normoxia condition; D: Statistical analysis of HIF-1α expression in VHL knockdown PANC-1 cells treated with CHX+TQ at 0 h, 1 h, 2 h and 3 h under normoxia conditions. a P < 0.01. CHX: Cycloheximide; VHL: von Hippel Lindau; sh-VHL: VHL knockdown; HIF-1α: Hypoxia-inducible factor-1α.

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Further validation of the effects of Thymoquinone on ubiquitination degradation of hypoxia-inducible factor-1αprotein. A: Western blot assay was performed to detect hypoxia-inducible factor-1α (HIF-1α) expression in von Hippel Lindau (VHL) knockdown PANC-1 cells treated with cycloheximide (CHX) at 0 h, 1 h, 2 h and 3 h under normoxia condition; B: Statistical analysis of HIF-1α protein expression in VHL knockdown PANC-1 cells treated with CHX at 0 h, 1 h, 2 h and 3 h under normoxia condition; C: Western blot assay was performed to detect HIF-1α expression in VHL knockdown PANC-1 cells treated with CHX + Thymoquinone (TQ) at 0 h, 1 h, 2 h and 3 h under normoxia condition; D: Statistical analysis of HIF-1α expression in VHL knockdown PANC-1 cells treated with CHX+TQ at 0 h, 1 h, 2 h and 3 h under normoxia conditions. a P < 0.01. CHX: Cycloheximide; VHL: von Hippel Lindau; sh-VHL: VHL knockdown; HIF-1α: Hypoxia-inducible factor-1α.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Biomarker Discovery, Ubiquitin Proteomics, Western Blot, Expressing, Knockdown

Full text summary. HIF-1α: Hypoxia-inducible factor-1α; HIF-1β: Hypoxia-inducible factor-1β; PI3K: Phosphatidyl inositol-4,5-bisphosphate-3-kinase; Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; 4E-BP1: Eukaryotic translation initiation factor 4E binding protein 1; eIF-4E: Eukaryotic translation initiation factor 4E; S6K: S6 kinase; Ras: Rat sarcoma; Raf: Rapidly accelerated fibrosarcoma; MAPK: Mitogen-activated protein kinases; ERK: Extracellular signal-regulated kinase; MEK: Mitogen extracellular signal-regulated kinas; MNK: MAP kinase interacting kinase; HRE: Hypoxia response elements; HSP90: Heat shock protein 90; pVHL: von Hippel-Lindau protein; Mdm2: Murine double-minute 2.

Journal: World Journal of Gastroenterology

Article Title: Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

doi: 10.3748/wjg.v30.i21.2793

Figure Lengend Snippet: Full text summary. HIF-1α: Hypoxia-inducible factor-1α; HIF-1β: Hypoxia-inducible factor-1β; PI3K: Phosphatidyl inositol-4,5-bisphosphate-3-kinase; Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; 4E-BP1: Eukaryotic translation initiation factor 4E binding protein 1; eIF-4E: Eukaryotic translation initiation factor 4E; S6K: S6 kinase; Ras: Rat sarcoma; Raf: Rapidly accelerated fibrosarcoma; MAPK: Mitogen-activated protein kinases; ERK: Extracellular signal-regulated kinase; MEK: Mitogen extracellular signal-regulated kinas; MNK: MAP kinase interacting kinase; HRE: Hypoxia response elements; HSP90: Heat shock protein 90; pVHL: von Hippel-Lindau protein; Mdm2: Murine double-minute 2.

Article Snippet: The following materials were used in this study: TQ (HY-D0803; MCE; New Jersey; United States); 26S proteasome inhibitor (HY-132598; MCE); geldanamycin (GA; HY-15230; MCE); cycloheximide (CHX) (S7418; SELLECK; Houston; United States); dimethyl sulfoxide (DMSO; Sigma; St. Louis; United States); fetal bovine serum (FBS; GIBCO; Invitrogen; Carlsbad; United States); Binding Buffer (XP2; Omega; Norcross; United States); trypsin-EDTA (GNM25200; Gino Biomedical Technology Co, Ltd; Hangzhou; China); Trypsin Solution without EDTA (C0205; Beyotime Biotech Co., Ltd; Shanghai; China); IP cell lysate (AS1003; Aspen Biotechnology Co., Ltd; Wuhan; China); SDS-PAGE gel preparation kit (AS1012; Aspen Biotechnology Co., Ltd; Wuhan; China); RPMI-1640 medium (GIBCO); Trizol (15596026; Ambion; Austin; United States); iScript® II Q RT SuperMix for quantitative real-time polymerase chain reaction (qPCR) (+gDNA wiper) (R233-01; VAZYME; Nangjing; China); HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01; VAZYME); SYBR Green Master Mix (Q111-02; VAZYME); Taq Plus DNA Polymerase (ET105-01; TIANGEN; Beijing; China); DL2000 DNA Marker (MD114-02; TIANGEN); primary antibodies: Anti-HIF1α (20960-1-AP; PROTEINTECH; Chicago; United States), anti-OH-HIF1α (3434T; CST; Danvers; United States), anti-phosphatidyl inositol-4,5-bisphosphate-3-kinase-p85α (PI3K-p85α; 60225-1-Ig; PROTEINTECH), anti-protein kinase B (Akt; 60203-2-Ig; PROTEINTECH), anti-mammalian target of rapamycin (mTOR; 66888-1-Ig; PROTEINTECH), anti-S6 kinase (S6K; 14485-1-AP; PROTEINTECH), anti-eukaryotic translation initiation factor 4E binding protein p70 S6 kinase (4E-BP1; 60246-1-Ig; PROTEINTECH), anti-eukaryotic translation initiation factor 4E (eIF-4E; 66655-1-Ig; PROTEINTECH), and anti-von Hippel Lindau (VHL) (16538-1-AP; PROTEINTECH); Cell counting kit-8 (CCK-8) kit (Beyotime Biotech Co., Ltd; Shanghai; China); Annexin V-FITC Apoptosis Detection Kit (AO2001-02P-G; Tianjin Sanjian Biotechnology Co., Ltd; Tianjin; China); phosphate-buffered saline (PBS, Gino Biomedical Technology Co, Ltd; Hangzhou; China); and tris-buffered saline (TBS; Gino Biomedical Technology Co, Ltd).

Techniques: Binding Assay

Figure 2 TCR-mediated virus-cell entry is highly specific and requires high-affinity interaction between the TCR and its cognate MHC/peptide complex. (a) Infection of EL4 cells by MV-eGFP, MV-T7, MV-m33, and MV-CD38 in the absence or presence of 5 106 M pOV8 (SIINFEKL) or SIYR peptide. Only the MV displaying the high-affinity m33 TCR infected these H-2b cells presenting the SIYR peptide. None of the viruses infected cells presenting the null peptide pOV8. Scale bar ¼ 300 mm. (b) Percentage of GFP positive after infection of SIYR or pOV8-loaded (3 106 M) EL4 (H-2b) and P815 (H-2d) cells with MV-m33. Infection of SIYR/EL4 cells by MV-m33 was efficiently inhibited by addition of anti-Kb antibody (50 mg/ml). Bars represent duplicate experiment.

Journal: Gene therapy

Article Title: Targeting virus entry and membrane fusion through specific peptide/MHC complexes using a high-affinity T-cell receptor.

doi: 10.1038/sj.gt.3302286

Figure Lengend Snippet: Figure 2 TCR-mediated virus-cell entry is highly specific and requires high-affinity interaction between the TCR and its cognate MHC/peptide complex. (a) Infection of EL4 cells by MV-eGFP, MV-T7, MV-m33, and MV-CD38 in the absence or presence of 5 106 M pOV8 (SIINFEKL) or SIYR peptide. Only the MV displaying the high-affinity m33 TCR infected these H-2b cells presenting the SIYR peptide. None of the viruses infected cells presenting the null peptide pOV8. Scale bar ¼ 300 mm. (b) Percentage of GFP positive after infection of SIYR or pOV8-loaded (3 106 M) EL4 (H-2b) and P815 (H-2d) cells with MV-m33. Infection of SIYR/EL4 cells by MV-m33 was efficiently inhibited by addition of anti-Kb antibody (50 mg/ml). Bars represent duplicate experiment.

Article Snippet: The EL4 T-cell lymphoma (H-2b), EL4 expressing chicken ovalbumin (E.G7.OVA, ATCC CRL 2113), EL4 expressing SIYRYYGL peptide (ETS-1), and P815 mastocytoma (H-2d, ATCC TIB-64) cells were grown in 10% FBS-DMEM.

Techniques: Virus, Infection

Figure 3 MV-m33 efficiently infected and caused extensive cell-to-cell fusion in EL4 cells stably expressing SIYR peptide (ETS-1), but not cells expressing chicken ovalbumin (EG7 OVA). (a) Cells were incubated with MV-eGFP, MV-T7 or MV-m33 at an MOI of 0.5 or 2.0 and photographed 48 h later. Only MV displaying the high-affinity scTCR (m33) was able to efficiently infect and cause extensive cell-to-cell fusion in ETS-1 cells. None of the viruses infected EL4 cells expressing a null peptide (EG7 OVA). Scale bar ¼ 300 mm. (b) Infection was also carried out in the presence of a fusion inhibitory peptide (FIP) that prevents cell-to-cell fusion. The percentage of single infected cells was analyzed by flow cytometry and expressed as the number of GFP-positive cells/ml of virus. (c) Infection of ETS-1 cells was correlated with the amount of MV-m33 virus added, and was inhibited more than 90% by anti-Kb antibody (50 mg/ ml). In contrast, the virus did not infect EG7 OVA cells expressing an irrelevant peptide/MHC complex (OVA/Kb).

Journal: Gene therapy

Article Title: Targeting virus entry and membrane fusion through specific peptide/MHC complexes using a high-affinity T-cell receptor.

doi: 10.1038/sj.gt.3302286

Figure Lengend Snippet: Figure 3 MV-m33 efficiently infected and caused extensive cell-to-cell fusion in EL4 cells stably expressing SIYR peptide (ETS-1), but not cells expressing chicken ovalbumin (EG7 OVA). (a) Cells were incubated with MV-eGFP, MV-T7 or MV-m33 at an MOI of 0.5 or 2.0 and photographed 48 h later. Only MV displaying the high-affinity scTCR (m33) was able to efficiently infect and cause extensive cell-to-cell fusion in ETS-1 cells. None of the viruses infected EL4 cells expressing a null peptide (EG7 OVA). Scale bar ¼ 300 mm. (b) Infection was also carried out in the presence of a fusion inhibitory peptide (FIP) that prevents cell-to-cell fusion. The percentage of single infected cells was analyzed by flow cytometry and expressed as the number of GFP-positive cells/ml of virus. (c) Infection of ETS-1 cells was correlated with the amount of MV-m33 virus added, and was inhibited more than 90% by anti-Kb antibody (50 mg/ ml). In contrast, the virus did not infect EG7 OVA cells expressing an irrelevant peptide/MHC complex (OVA/Kb).

Article Snippet: The EL4 T-cell lymphoma (H-2b), EL4 expressing chicken ovalbumin (E.G7.OVA, ATCC CRL 2113), EL4 expressing SIYRYYGL peptide (ETS-1), and P815 mastocytoma (H-2d, ATCC TIB-64) cells were grown in 10% FBS-DMEM.

Techniques: Infection, Stable Transfection, Expressing, Incubation, Cytometry, Virus

Figure 4 Infection of EL4 cells by MV-m33 is dependent on the dose of SIYR peptide. (a) EL4 cells were loaded with increasing concentrations of SIYR peptide and infected with MV-m33 at MOIs of 2.0, 5.0, and 10.0. The percentage of GFP-positive cells was determined 72 h post infection by flow cytometry. (b) Photograph of infected EL4 cells loaded with 3 108–3 106 M SIYR peptide. (c) Number of peptide/MHC complexes on EL4 cells after loading with increasing concentrations of SIYR peptide. The number of peptide/MHC complexes present on ETS-1 cells is also shown (arrow).

Journal: Gene therapy

Article Title: Targeting virus entry and membrane fusion through specific peptide/MHC complexes using a high-affinity T-cell receptor.

doi: 10.1038/sj.gt.3302286

Figure Lengend Snippet: Figure 4 Infection of EL4 cells by MV-m33 is dependent on the dose of SIYR peptide. (a) EL4 cells were loaded with increasing concentrations of SIYR peptide and infected with MV-m33 at MOIs of 2.0, 5.0, and 10.0. The percentage of GFP-positive cells was determined 72 h post infection by flow cytometry. (b) Photograph of infected EL4 cells loaded with 3 108–3 106 M SIYR peptide. (c) Number of peptide/MHC complexes on EL4 cells after loading with increasing concentrations of SIYR peptide. The number of peptide/MHC complexes present on ETS-1 cells is also shown (arrow).

Article Snippet: The EL4 T-cell lymphoma (H-2b), EL4 expressing chicken ovalbumin (E.G7.OVA, ATCC CRL 2113), EL4 expressing SIYRYYGL peptide (ETS-1), and P815 mastocytoma (H-2d, ATCC TIB-64) cells were grown in 10% FBS-DMEM.

Techniques: Infection, Cytometry

FIGURE 2. Characterization of recombinant GDF6, GDF7, and BMP10. A, immuno- blotting analyses of recombinant GDF6 and GDF7. Recombinant proteins were analyzed under reduced or nonreduced conditions following blotting with specific antibodies. B, biochemical properties of purified recombinant human BMP10. BMP10 with an N-termi- nal His6 tag was purified from conditioned media of 293T cells using metal chelate chro- matography. BMP10 was detected by Coomassie Blue staining (lanes 1–3) and immuno- blotting using antibodies against polyhistidine His6 (lanes 4 and 5) under nonreduced or reduced conditions. Some samples were treated with peptide N-glycosidase F (PNGase) to remove N-linked carbohydrate side chains.

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 2. Characterization of recombinant GDF6, GDF7, and BMP10. A, immuno- blotting analyses of recombinant GDF6 and GDF7. Recombinant proteins were analyzed under reduced or nonreduced conditions following blotting with specific antibodies. B, biochemical properties of purified recombinant human BMP10. BMP10 with an N-termi- nal His6 tag was purified from conditioned media of 293T cells using metal chelate chro- matography. BMP10 was detected by Coomassie Blue staining (lanes 1–3) and immuno- blotting using antibodies against polyhistidine His6 (lanes 4 and 5) under nonreduced or reduced conditions. Some samples were treated with peptide N-glycosidase F (PNGase) to remove N-linked carbohydrate side chains.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Recombinant, Purification, Staining

FIGURE 3. Treatment with GDF6, GDF7, and BMP10 stimulated the Smad1/5/8 pathway but not the Smad2/3 pathway in mouse MC3T3 cells. Cells were transiently transfected with the BMP-responsive reporter BRE (A) or the TGF/ac- tivin-responsive CAGA reporter (B). At 48 h after transfection, cells were incubated for 20 h in the absence(Ct)orpresenceofGDF6(10nM),GDF7(10 nM), or BMP10 (3 nM). Cells treated with BMP2 (0.1 nM), BMP7 (3 nM), and TGF (0.1 nM) served as pos- itive controls. C, cells were transfected with the BRE reporter and treated with increasing doses of GDF6, GDF7, or BMP10. The relative luciferase activity was normalized based on the -galacto- sidase activity to correct for variations in transfec- tion efficiency. Results are presented as the mean S.E. D, immunoblotting analysis of Smad1 and Smad2 phosphorylation was performed using cell extracts following treatment of MC3T3 cells with BMP2 (0.1 nM), TGF (0.1 nM), GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). Treatment with GDF6, GDF7, or BMP10 induced the phosphoryla- tion of Smad1 (top) but not Smad2 (Bottom). BMP2 and TGF served as positive controls. Arrows indi- cate the immunoreactive bands of phosphoryl- ated Smad proteins. Ct, control.

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 3. Treatment with GDF6, GDF7, and BMP10 stimulated the Smad1/5/8 pathway but not the Smad2/3 pathway in mouse MC3T3 cells. Cells were transiently transfected with the BMP-responsive reporter BRE (A) or the TGF/ac- tivin-responsive CAGA reporter (B). At 48 h after transfection, cells were incubated for 20 h in the absence(Ct)orpresenceofGDF6(10nM),GDF7(10 nM), or BMP10 (3 nM). Cells treated with BMP2 (0.1 nM), BMP7 (3 nM), and TGF (0.1 nM) served as pos- itive controls. C, cells were transfected with the BRE reporter and treated with increasing doses of GDF6, GDF7, or BMP10. The relative luciferase activity was normalized based on the -galacto- sidase activity to correct for variations in transfec- tion efficiency. Results are presented as the mean S.E. D, immunoblotting analysis of Smad1 and Smad2 phosphorylation was performed using cell extracts following treatment of MC3T3 cells with BMP2 (0.1 nM), TGF (0.1 nM), GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). Treatment with GDF6, GDF7, or BMP10 induced the phosphoryla- tion of Smad1 (top) but not Smad2 (Bottom). BMP2 and TGF served as positive controls. Arrows indi- cate the immunoreactive bands of phosphoryl- ated Smad proteins. Ct, control.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Incubation, Luciferase, Activity Assay, Western Blot, Phospho-proteomics, Control

FIGURE 4. Antagonistic effects of inhibitory Smad proteins on the stimulation of the BRE promoter by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of plasmids encoding the inhibitory Smad6 or Smad7. After transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), BMP10 (3 nM), or BMP2 (0.1 nM), before determina- tion of luciferase activity. The relative luciferase activity was normalized based on the -galactosidase activity to correct for variations in transfection efficiency. Results are presented as the mean S.E.

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 4. Antagonistic effects of inhibitory Smad proteins on the stimulation of the BRE promoter by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of plasmids encoding the inhibitory Smad6 or Smad7. After transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), BMP10 (3 nM), or BMP2 (0.1 nM), before determina- tion of luciferase activity. The relative luciferase activity was normalized based on the -galactosidase activity to correct for variations in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay

FIGURE 5. Overexpression of ALK3 or ALK6 confers ligand signaling by GDF6, GDF7, or BMP10. A, COS7 cells were transfected with 500 ng of the BRE reporter construct together with 30 ng of different ALK plasmids or increasing amounts of the ALK3 or ALK6 plasmid. After 5 h of incubation, cells were treated with GDF6 (1 nM), GDF7 (1 nM), or BMP10 (3 nM) for 24 h. The relative luciferase activity was normalized based on the -galactosidase activity. Results are presented as the mean S.E. B, confirmation of the overexpression of different human ALK receptor transcripts in transfected cells using semi-quantitative PCR. , transfection with the empty vector; , transfection with individual ALK plasmid. In select cases, the endogenous monkey transcripts were amplified in COS7 cells because of sequence conservation.

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 5. Overexpression of ALK3 or ALK6 confers ligand signaling by GDF6, GDF7, or BMP10. A, COS7 cells were transfected with 500 ng of the BRE reporter construct together with 30 ng of different ALK plasmids or increasing amounts of the ALK3 or ALK6 plasmid. After 5 h of incubation, cells were treated with GDF6 (1 nM), GDF7 (1 nM), or BMP10 (3 nM) for 24 h. The relative luciferase activity was normalized based on the -galactosidase activity. Results are presented as the mean S.E. B, confirmation of the overexpression of different human ALK receptor transcripts in transfected cells using semi-quantitative PCR. , transfection with the empty vector; , transfection with individual ALK plasmid. In select cases, the endogenous monkey transcripts were amplified in COS7 cells because of sequence conservation.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, Transfection, Construct, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Amplification, Sequencing

FIGURE 7. Overexpression of the ALK3 shRNA suppressed the stimulation of BRE promoter activities by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the ALK3 shRNA or control shRNA. Two days after transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). As a negative control, cells were transfected with the ALK3 shRNA before treatment with TGF (0.1 nM). The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 7. Overexpression of the ALK3 shRNA suppressed the stimulation of BRE promoter activities by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the ALK3 shRNA or control shRNA. Two days after transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). As a negative control, cells were transfected with the ALK3 shRNA before treatment with TGF (0.1 nM). The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, shRNA, Transfection, Plasmid Preparation, Control, Incubation, Negative Control, Activity Assay

FIGURE 8. Overexpression of BMPRII shRNA or ActRIIA shRNA blocked the stimula- tion of the BRE promoter activity by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the BMPRII, ActRIIA, or control shRNA. As a negative control, cells were transfected with the BMPRII shRNA or ActRIIA shRNA before treatment with TGF. The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 8. Overexpression of BMPRII shRNA or ActRIIA shRNA blocked the stimula- tion of the BRE promoter activity by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the BMPRII, ActRIIA, or control shRNA. As a negative control, cells were transfected with the BMPRII shRNA or ActRIIA shRNA before treatment with TGF. The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, shRNA, Activity Assay, Transfection, Plasmid Preparation, Control, Negative Control