cdk2 transcript knockdown Search Results


vascular endothelial growth factor  (Proteintech)
96
Proteintech vascular endothelial growth factor
After 1-week acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): control (saline), NE (0.125 mg/kg), VEN (10 mg/kg), and drug combination [NE (0.125 mg/kg) + VEN (10 mg/kg)], and administered intraperitoneally with each drug or combination every day. One week later, 1 × 10 6 CT26 cells were injected subcutaneously into the right oxters of the mice. Furthermore, mice of each group were treated with the above drug or drug combination for additional 17 days. A The volumes of tumor xenografts were measured and revealed (upper left). On day 17, mice were sacrificed, and tumor xenografts were removed. The gross morphology, tumor volume, and tumor weight of xenografts were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining and CD34 immunohistochemistry (IHC) analysis of the xenograft sections were performed. Microvessel density (MVD) was counted based on CD34 IHC. C The xenograft lysates were collected, and the expressions of CD34, <t>VEGF,</t> and NET were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01 in A , B , C .
Vascular Endothelial Growth Factor, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cdk2 transcript knockdown  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology cdk2 transcript knockdown
After 1-week acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): control (saline), NE (0.125 mg/kg), VEN (10 mg/kg), and drug combination [NE (0.125 mg/kg) + VEN (10 mg/kg)], and administered intraperitoneally with each drug or combination every day. One week later, 1 × 10 6 CT26 cells were injected subcutaneously into the right oxters of the mice. Furthermore, mice of each group were treated with the above drug or drug combination for additional 17 days. A The volumes of tumor xenografts were measured and revealed (upper left). On day 17, mice were sacrificed, and tumor xenografts were removed. The gross morphology, tumor volume, and tumor weight of xenografts were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining and CD34 immunohistochemistry (IHC) analysis of the xenograft sections were performed. Microvessel density (MVD) was counted based on CD34 IHC. C The xenograft lysates were collected, and the expressions of CD34, <t>VEGF,</t> and NET were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01 in A , B , C .
Cdk2 Transcript Knockdown, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal ab against cdk2  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology rabbit polyclonal ab against cdk2
Fig. 3. δEF1 concurrently up-regulates the expressions of <t>CDK2</t> and CDK4 while promoting cell proliferation of MDA-MB-231 cells. (A) δEF1-induced up-regulation of CDK2 and CDK4 mRNA levels in MDA-MB-231 cells were verified by quantitative RT-PCR. GAPDH was used to normalize the CDK2 and CDK4 levels. Data represent three independent experiments. (B) δEF1-specific siRNA plasmid (si-δEF1) was introduced into MDA-MB-231 cells using transient transfection. Control cells were treated with a scrambled siRNA. The efficiency of δEF1 mRNA knockdown was examined by RT-PCR. The inhibition of CDK2 and CDK4 mRNA levels were determined at different time points (0, 24, and 48 h) after transfection, using quantitative RT- PCR. GAPDH was used to normalize the CKD2 and CDK4 levels. SIP1 was used as an off-set control of siRNA interference. Data represent three independent experiments. (C) δEF1-induced up-regulation of CDK2 and CDK4 protein levels in MDA-MB-231 cells was verified by western blotting. Actin was used to normalize the CDK2 and CDK4 levels.
Rabbit Polyclonal Ab Against Cdk2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cdk2 shrna m lentiviral particles  (Santa Cruz Biotechnology)
85
Santa Cruz Biotechnology cdk2 shrna m lentiviral particles
A. Spontaneous colony formation in AMPKα1-KO MEFs. Wild type (WT), AMPKα1-KO, and AMPKα2-KO MEFs (1 × 10 5 cells/mL) were seeded and cultured on 6-well plates. Culture medium was changed every 2 days for 3 weeks. (Upper) Representative images showing colony formation of MEFs. (Bottom) Quantification of colony formation. n =5, * P <0.001 versus WT. B. Anchorage-independent cell growth assay (soft agar assay) of MEFs. (Upper) Representative images for colony formation. Scale bar =500 μm. (Bottom) Quantification of colony formation. n =10, * P <0.001 versus WT. C. Phosphorylated <t>CDK2</t> at The-160 (pCDK2-T160) and CDK2 are upregulated in AMPKα1-KO MEFs. pCDK2-T160 and <t>CDK2</t> <t>protein</t> in WT, AMPKα1-KO, and AMPKα2-KO MEFs were analyzed by Western blot (top). Quantification of pCDK2 and CDK2 data (bottom). n =4, * P <0.01 versus WT; † P <0.05 versus WT. D. Diminished anchorage-independent growth of AMPKα1-KO MEFs following CDK2 knockdown by <t>shRNA.</t> Representative images are shown. Scale bar =500 μm. E. Representative Western blot data indicate CDK2 knockdown by shRNA (top). Quantification of anchorage-independent MEF growth (bottom). n =4, * P <0.01 versus WT/control shRNA; † P <0.01 versus α1-KO/control shRNA.
Cdk2 Shrna M Lentiviral Particles, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


After 1-week acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): control (saline), NE (0.125 mg/kg), VEN (10 mg/kg), and drug combination [NE (0.125 mg/kg) + VEN (10 mg/kg)], and administered intraperitoneally with each drug or combination every day. One week later, 1 × 10 6 CT26 cells were injected subcutaneously into the right oxters of the mice. Furthermore, mice of each group were treated with the above drug or drug combination for additional 17 days. A The volumes of tumor xenografts were measured and revealed (upper left). On day 17, mice were sacrificed, and tumor xenografts were removed. The gross morphology, tumor volume, and tumor weight of xenografts were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining and CD34 immunohistochemistry (IHC) analysis of the xenograft sections were performed. Microvessel density (MVD) was counted based on CD34 IHC. C The xenograft lysates were collected, and the expressions of CD34, VEGF, and NET were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01 in A , B , C .

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: After 1-week acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): control (saline), NE (0.125 mg/kg), VEN (10 mg/kg), and drug combination [NE (0.125 mg/kg) + VEN (10 mg/kg)], and administered intraperitoneally with each drug or combination every day. One week later, 1 × 10 6 CT26 cells were injected subcutaneously into the right oxters of the mice. Furthermore, mice of each group were treated with the above drug or drug combination for additional 17 days. A The volumes of tumor xenografts were measured and revealed (upper left). On day 17, mice were sacrificed, and tumor xenografts were removed. The gross morphology, tumor volume, and tumor weight of xenografts were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining and CD34 immunohistochemistry (IHC) analysis of the xenograft sections were performed. Microvessel density (MVD) was counted based on CD34 IHC. C The xenograft lysates were collected, and the expressions of CD34, VEGF, and NET were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01 in A , B , C .

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Control, Saline, Injection, Staining, Immunohistochemistry, Western Blot

Colon cancer cells (CT26, HCT116, and SW480) were treated with control, VEN (10 μM), NE (10 μM), and drug combination [NE (10 μM) + VEN (10 μM)] for 24 h and 48 h separately. A The effects of NE/VEN on colon cancer cell proliferation were determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. B Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was performed and revealed an increase in the VEGF mRNA with NE treatment, while VEN inhibited the NE-increased VEGF mRNA in colon cancer cells. C Western blotting was performed and revealed increases in VEGF protein and Akt activation with NE treatment, while VEN inhibited these NE-induced changes in colon cancer cells. The band intensities of VEGF, pAkt, and Akt relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified and normalized to the Ctrl. group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01, or *** P < 0.001 in ( A ) and ( B ).

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: Colon cancer cells (CT26, HCT116, and SW480) were treated with control, VEN (10 μM), NE (10 μM), and drug combination [NE (10 μM) + VEN (10 μM)] for 24 h and 48 h separately. A The effects of NE/VEN on colon cancer cell proliferation were determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. B Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was performed and revealed an increase in the VEGF mRNA with NE treatment, while VEN inhibited the NE-increased VEGF mRNA in colon cancer cells. C Western blotting was performed and revealed increases in VEGF protein and Akt activation with NE treatment, while VEN inhibited these NE-induced changes in colon cancer cells. The band intensities of VEGF, pAkt, and Akt relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified and normalized to the Ctrl. group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01, or *** P < 0.001 in ( A ) and ( B ).

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Control, MTT Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Activation Assay

Colon cancer cells were treated with control, venlafaxine (VEN) (10 μM), NE (10 μM), and drug combination [NE (10 μM) + VEN (10 μM)]. A Western blotting and B Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay were used to detect the changes in NET protein and mRNA. The band intensities of NET relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified and normalized to the Ctrl. group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . C Colon cancer cells were transfected with siNET, sihNET, or siNC separately to knock down the expression of NET. After 24 h, they were treated with 10 μM of NE and incubated further for 24 h and 48 h. The effects of NE/siNET on colon cancer cell proliferation were determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. D Western blotting and E qRT-PCR assay were performed and revealed the knockdown of NET inhibited the NE-increased VEGF protein,VEGF mRNA, and Akt activation in colon cancer cells. The band intensities of VEGF, pAkt, and Akt relative to GAPDH were quantified and normalized to the siNC group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01, or *** P < 0.001 in ( B ), ( C ) and ( E ).

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: Colon cancer cells were treated with control, venlafaxine (VEN) (10 μM), NE (10 μM), and drug combination [NE (10 μM) + VEN (10 μM)]. A Western blotting and B Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay were used to detect the changes in NET protein and mRNA. The band intensities of NET relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified and normalized to the Ctrl. group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . C Colon cancer cells were transfected with siNET, sihNET, or siNC separately to knock down the expression of NET. After 24 h, they were treated with 10 μM of NE and incubated further for 24 h and 48 h. The effects of NE/siNET on colon cancer cell proliferation were determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. D Western blotting and E qRT-PCR assay were performed and revealed the knockdown of NET inhibited the NE-increased VEGF protein,VEGF mRNA, and Akt activation in colon cancer cells. The band intensities of VEGF, pAkt, and Akt relative to GAPDH were quantified and normalized to the siNC group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01, or *** P < 0.001 in ( B ), ( C ) and ( E ).

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Knockdown, Expressing, Incubation, MTT Assay, Activation Assay

A CT26 cells were transfected with plasmids of ovCtrl., ovPPP2R1A, or ovNET separately. Twenty-four hours later, they were treated with NE or not and incubated further for 48 h. Western blotting was used to detect the changes in PPP2R1A, pAkt, Akt, and VEGF proteins. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . B CT26 cells were transfected with short interfering RNAs of siNC, siPPP2R1A-1, or siPPP2R1A-2 separately. Twenty-four hours later, they were treated with venlafaxine (VEN), NE, and NE + VEN, or not and incubated further for 48 h. Western blotting was used to detect the changes in PPP2R1A, pAkt, Akt, and VEGF proteins. The band intensities of PPP2R1A, pAkt, Akt, and VEGF relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified and normalized to the ovCtrl./siNC group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . Experiments were repeated three independent times with reproducible results.

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: A CT26 cells were transfected with plasmids of ovCtrl., ovPPP2R1A, or ovNET separately. Twenty-four hours later, they were treated with NE or not and incubated further for 48 h. Western blotting was used to detect the changes in PPP2R1A, pAkt, Akt, and VEGF proteins. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . B CT26 cells were transfected with short interfering RNAs of siNC, siPPP2R1A-1, or siPPP2R1A-2 separately. Twenty-four hours later, they were treated with venlafaxine (VEN), NE, and NE + VEN, or not and incubated further for 48 h. Western blotting was used to detect the changes in PPP2R1A, pAkt, Akt, and VEGF proteins. The band intensities of PPP2R1A, pAkt, Akt, and VEGF relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified and normalized to the ovCtrl./siNC group. The quantification graphs of normalized band intensities of different replicates were shown in Fig. . Experiments were repeated three independent times with reproducible results.

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Transfection, Incubation, Western Blot

A Following acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): CT26-Lv-shCtrl.+saline, CT26-Lv-shNET-3+saline, CT26-Lv-shCtrl.+NE, and CT26-Lv-shNET-3+NE. Male BALB/c mice were intraperitoneally administered with saline or NE (0.125 mg/kg) daily. One week later, 1 × 10 6 stable cells (CT26-Lv-shNET-3 or CT26-Lv-shCtrl.) were injected subcutaneously into the mice separately. In addition, mice of each group were treated with saline or NE for 17 days. Volumes of tumor xenografts were measured (upper left). On day 17 following cell inoculation, mice were scarified, and tumor xenografts were removed. The gross morphology of tumors, tumor volume, and tumor weight of each group were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining of the xenograft sections and CD34 immunohistochemical analysis was performed. C Xenograft lysates were collected, and the expression levels of PPP2R1A, pAkt, Akt, VEGF, and CD34 were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01, or *** P < 0.001 in A and C .

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: A Following acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): CT26-Lv-shCtrl.+saline, CT26-Lv-shNET-3+saline, CT26-Lv-shCtrl.+NE, and CT26-Lv-shNET-3+NE. Male BALB/c mice were intraperitoneally administered with saline or NE (0.125 mg/kg) daily. One week later, 1 × 10 6 stable cells (CT26-Lv-shNET-3 or CT26-Lv-shCtrl.) were injected subcutaneously into the mice separately. In addition, mice of each group were treated with saline or NE for 17 days. Volumes of tumor xenografts were measured (upper left). On day 17 following cell inoculation, mice were scarified, and tumor xenografts were removed. The gross morphology of tumors, tumor volume, and tumor weight of each group were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining of the xenograft sections and CD34 immunohistochemical analysis was performed. C Xenograft lysates were collected, and the expression levels of PPP2R1A, pAkt, Akt, VEGF, and CD34 were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01, or *** P < 0.001 in A and C .

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Saline, Injection, Staining, Immunohistochemical staining, Expressing, Western Blot

By binding with beta-adrenergic receptor (b-AR), norepinephrine (NE) induces Akt activation, VEGF expression, cell proliferation, angiogenesis, and cancer progression in human colorectal cancer (CRC) cells. NE also increases the expression of NE transporter (NET). Venlafaxine (VEN) can antagonize the above effects of NE by inhibiting the NE-increased NET expression, which is related to the interaction of NET and protein phosphatase 2 scaffold subunit alpha (PPP2R1A).

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: By binding with beta-adrenergic receptor (b-AR), norepinephrine (NE) induces Akt activation, VEGF expression, cell proliferation, angiogenesis, and cancer progression in human colorectal cancer (CRC) cells. NE also increases the expression of NE transporter (NET). Venlafaxine (VEN) can antagonize the above effects of NE by inhibiting the NE-increased NET expression, which is related to the interaction of NET and protein phosphatase 2 scaffold subunit alpha (PPP2R1A).

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Binding Assay, Activation Assay, Expressing

Fig. 3. δEF1 concurrently up-regulates the expressions of CDK2 and CDK4 while promoting cell proliferation of MDA-MB-231 cells. (A) δEF1-induced up-regulation of CDK2 and CDK4 mRNA levels in MDA-MB-231 cells were verified by quantitative RT-PCR. GAPDH was used to normalize the CDK2 and CDK4 levels. Data represent three independent experiments. (B) δEF1-specific siRNA plasmid (si-δEF1) was introduced into MDA-MB-231 cells using transient transfection. Control cells were treated with a scrambled siRNA. The efficiency of δEF1 mRNA knockdown was examined by RT-PCR. The inhibition of CDK2 and CDK4 mRNA levels were determined at different time points (0, 24, and 48 h) after transfection, using quantitative RT- PCR. GAPDH was used to normalize the CKD2 and CDK4 levels. SIP1 was used as an off-set control of siRNA interference. Data represent three independent experiments. (C) δEF1-induced up-regulation of CDK2 and CDK4 protein levels in MDA-MB-231 cells was verified by western blotting. Actin was used to normalize the CDK2 and CDK4 levels.

Journal: Biochimica et biophysica acta

Article Title: DeltaEF1 promotes breast cancer cell proliferation through down-regulating p21 expression.

doi: 10.1016/j.bbadis.2009.12.002

Figure Lengend Snippet: Fig. 3. δEF1 concurrently up-regulates the expressions of CDK2 and CDK4 while promoting cell proliferation of MDA-MB-231 cells. (A) δEF1-induced up-regulation of CDK2 and CDK4 mRNA levels in MDA-MB-231 cells were verified by quantitative RT-PCR. GAPDH was used to normalize the CDK2 and CDK4 levels. Data represent three independent experiments. (B) δEF1-specific siRNA plasmid (si-δEF1) was introduced into MDA-MB-231 cells using transient transfection. Control cells were treated with a scrambled siRNA. The efficiency of δEF1 mRNA knockdown was examined by RT-PCR. The inhibition of CDK2 and CDK4 mRNA levels were determined at different time points (0, 24, and 48 h) after transfection, using quantitative RT- PCR. GAPDH was used to normalize the CKD2 and CDK4 levels. SIP1 was used as an off-set control of siRNA interference. Data represent three independent experiments. (C) δEF1-induced up-regulation of CDK2 and CDK4 protein levels in MDA-MB-231 cells was verified by western blotting. Actin was used to normalize the CDK2 and CDK4 levels.

Article Snippet: The following antibodies (Abs) were used: goat polyclonal Ab against the N-terminal epitope of δEF1 (ZEB-E20, Santa Cruz); rabbit polyclonal Ab against CDK2 (sc-163, Santa Cruz); rabbit polyclonal Ab against CDK4 (sc-260, Santa Cruz); mouse monoclonal Ab against p21 (sc6246, Santa Cruz); mouse monoclonal Ab against p27 (sc-56454, Santa Cruz); mouse monoclonal Ab against Actin (A-4700, Sigma).

Techniques: Quantitative RT-PCR, Plasmid Preparation, Transfection, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Inhibition, Western Blot

Fig. 5. δEF1 overexpression increases its recruitment to endogenous p21 promoter in an E2-box-dependent manner. (A) Association of δEF1 with the proximal human p21 promoter was analyzed by CHIP analyses in MDA-MB-231 cells, using polyclonal antibody against δEF1 or unrelated IgG antibody. Amplified human p21 promoter fragment of E2 box containing sequence is shown. Amount of DNA in input confirms equal loading of chromatin. (B) δEF1 transfection to MDA-MB-231 cells significantly enhanced its recruitment to endogenous p21 promoter by quantitative CHIP assay. ⁎pb0.05 in unpaired Student t test when compared with vector alone. Data represent three independent experiments.

Journal: Biochimica et biophysica acta

Article Title: DeltaEF1 promotes breast cancer cell proliferation through down-regulating p21 expression.

doi: 10.1016/j.bbadis.2009.12.002

Figure Lengend Snippet: Fig. 5. δEF1 overexpression increases its recruitment to endogenous p21 promoter in an E2-box-dependent manner. (A) Association of δEF1 with the proximal human p21 promoter was analyzed by CHIP analyses in MDA-MB-231 cells, using polyclonal antibody against δEF1 or unrelated IgG antibody. Amplified human p21 promoter fragment of E2 box containing sequence is shown. Amount of DNA in input confirms equal loading of chromatin. (B) δEF1 transfection to MDA-MB-231 cells significantly enhanced its recruitment to endogenous p21 promoter by quantitative CHIP assay. ⁎pb0.05 in unpaired Student t test when compared with vector alone. Data represent three independent experiments.

Article Snippet: The following antibodies (Abs) were used: goat polyclonal Ab against the N-terminal epitope of δEF1 (ZEB-E20, Santa Cruz); rabbit polyclonal Ab against CDK2 (sc-163, Santa Cruz); rabbit polyclonal Ab against CDK4 (sc-260, Santa Cruz); mouse monoclonal Ab against p21 (sc6246, Santa Cruz); mouse monoclonal Ab against p27 (sc-56454, Santa Cruz); mouse monoclonal Ab against Actin (A-4700, Sigma).

Techniques: Over Expression, Sequencing, Transfection, Plasmid Preparation

A. Spontaneous colony formation in AMPKα1-KO MEFs. Wild type (WT), AMPKα1-KO, and AMPKα2-KO MEFs (1 × 10 5 cells/mL) were seeded and cultured on 6-well plates. Culture medium was changed every 2 days for 3 weeks. (Upper) Representative images showing colony formation of MEFs. (Bottom) Quantification of colony formation. n =5, * P <0.001 versus WT. B. Anchorage-independent cell growth assay (soft agar assay) of MEFs. (Upper) Representative images for colony formation. Scale bar =500 μm. (Bottom) Quantification of colony formation. n =10, * P <0.001 versus WT. C. Phosphorylated CDK2 at The-160 (pCDK2-T160) and CDK2 are upregulated in AMPKα1-KO MEFs. pCDK2-T160 and CDK2 protein in WT, AMPKα1-KO, and AMPKα2-KO MEFs were analyzed by Western blot (top). Quantification of pCDK2 and CDK2 data (bottom). n =4, * P <0.01 versus WT; † P <0.05 versus WT. D. Diminished anchorage-independent growth of AMPKα1-KO MEFs following CDK2 knockdown by shRNA. Representative images are shown. Scale bar =500 μm. E. Representative Western blot data indicate CDK2 knockdown by shRNA (top). Quantification of anchorage-independent MEF growth (bottom). n =4, * P <0.01 versus WT/control shRNA; † P <0.01 versus α1-KO/control shRNA.

Journal: Oncotarget

Article Title: AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2

doi: 10.18632/oncotarget.10687

Figure Lengend Snippet: A. Spontaneous colony formation in AMPKα1-KO MEFs. Wild type (WT), AMPKα1-KO, and AMPKα2-KO MEFs (1 × 10 5 cells/mL) were seeded and cultured on 6-well plates. Culture medium was changed every 2 days for 3 weeks. (Upper) Representative images showing colony formation of MEFs. (Bottom) Quantification of colony formation. n =5, * P <0.001 versus WT. B. Anchorage-independent cell growth assay (soft agar assay) of MEFs. (Upper) Representative images for colony formation. Scale bar =500 μm. (Bottom) Quantification of colony formation. n =10, * P <0.001 versus WT. C. Phosphorylated CDK2 at The-160 (pCDK2-T160) and CDK2 are upregulated in AMPKα1-KO MEFs. pCDK2-T160 and CDK2 protein in WT, AMPKα1-KO, and AMPKα2-KO MEFs were analyzed by Western blot (top). Quantification of pCDK2 and CDK2 data (bottom). n =4, * P <0.01 versus WT; † P <0.05 versus WT. D. Diminished anchorage-independent growth of AMPKα1-KO MEFs following CDK2 knockdown by shRNA. Representative images are shown. Scale bar =500 μm. E. Representative Western blot data indicate CDK2 knockdown by shRNA (top). Quantification of anchorage-independent MEF growth (bottom). n =4, * P <0.01 versus WT/control shRNA; † P <0.01 versus α1-KO/control shRNA.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): rabbit anti-CDK2 (2546), rabbit anti-pCDK2-T160 (2561), rabbit anti-pp100-S866/870 (4810), rabbit anti-pIĸKα/β (2078), rabbit anti-IĸKα (2682), rabbit anti-NF-ĸB2 p100/p52 (4882), rabbit anti-NIK (4994), rabbit anti-β-TrCP (D13F10) (4394), and rabbit anti-PCNA (13110). β-TrCP siRNA (sc-37179), p52 siRNA (m) (sc-36043), CDK2 shRNA (m) lentiviral particles (sc-29260-V), NFκB p52 shRNA (m) lentiviral particles (sc-36043-V), goat anti-pNIK-T559 (sc-12957), rabbit anti-Epo (sc-7956), mouse anti-GAPDH (sc-32233), mouse anti-β-actin (sc-47778), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, Growth Assay, Soft Agar Assay, Western Blot, Knockdown, shRNA, Control

A. Upregulation of CDK2 transcription in AMPKα1-KO MEFs. CDK2 mRNA levels were measured by quantitative reverse transcription polymerase chain reaction in WT and AMPKα1-KO MEFs. n =5, * P <0.01 versus WT. B. p52 is responsible for CDK2 elevation in AMPKα1-KO MEFs. MEFs were transfected with either control siRNA or p52 siRNA (100 nmol/L) for 72 hours. Representative blot from three independent experiments (top). Quantification of Western blot data (bottom). n =3, * P <0.01 versus WT/control siRNA; † P <0.05 versus α1-KO/control siRNA. C. Chromatin immunoprecipitation (ChIP) analysis of the CDK2 gene. MEF chromatin from WT and AMPKα1-KO mice was immunoprecipitated with anti-p52 or rabbit IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for mouse CDK2 promoter. This result is representative of four independent experiments. n =4, * P <0.05 versus WT. D. p52 knockdown by shRNA diminishes anchorage-independent growth of AMPKα1-KO MEFs. Representative images are shown. Scale bar =500 μm. E. Quantification of anchorage-independent MEF growth. Data are mean ± SD, n =5, * P <0.01 versus WT/control shRNA; † P <0.01 versus α1-KO/control shRNA.

Journal: Oncotarget

Article Title: AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2

doi: 10.18632/oncotarget.10687

Figure Lengend Snippet: A. Upregulation of CDK2 transcription in AMPKα1-KO MEFs. CDK2 mRNA levels were measured by quantitative reverse transcription polymerase chain reaction in WT and AMPKα1-KO MEFs. n =5, * P <0.01 versus WT. B. p52 is responsible for CDK2 elevation in AMPKα1-KO MEFs. MEFs were transfected with either control siRNA or p52 siRNA (100 nmol/L) for 72 hours. Representative blot from three independent experiments (top). Quantification of Western blot data (bottom). n =3, * P <0.01 versus WT/control siRNA; † P <0.05 versus α1-KO/control siRNA. C. Chromatin immunoprecipitation (ChIP) analysis of the CDK2 gene. MEF chromatin from WT and AMPKα1-KO mice was immunoprecipitated with anti-p52 or rabbit IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for mouse CDK2 promoter. This result is representative of four independent experiments. n =4, * P <0.05 versus WT. D. p52 knockdown by shRNA diminishes anchorage-independent growth of AMPKα1-KO MEFs. Representative images are shown. Scale bar =500 μm. E. Quantification of anchorage-independent MEF growth. Data are mean ± SD, n =5, * P <0.01 versus WT/control shRNA; † P <0.01 versus α1-KO/control shRNA.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): rabbit anti-CDK2 (2546), rabbit anti-pCDK2-T160 (2561), rabbit anti-pp100-S866/870 (4810), rabbit anti-pIĸKα/β (2078), rabbit anti-IĸKα (2682), rabbit anti-NF-ĸB2 p100/p52 (4882), rabbit anti-NIK (4994), rabbit anti-β-TrCP (D13F10) (4394), and rabbit anti-PCNA (13110). β-TrCP siRNA (sc-37179), p52 siRNA (m) (sc-36043), CDK2 shRNA (m) lentiviral particles (sc-29260-V), NFκB p52 shRNA (m) lentiviral particles (sc-36043-V), goat anti-pNIK-T559 (sc-12957), rabbit anti-Epo (sc-7956), mouse anti-GAPDH (sc-32233), mouse anti-β-actin (sc-47778), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Transfection, Control, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Amplification, Knockdown, shRNA

A. Plasma concentrations of Epo in nude mice inoculated with WT or AMPKα1-KO MEFs. n =10-13 per group; * P <0.01 versus WT. B. Epo upregulation in inoculated AMPKα1-KO MEFs in nude mice. Implanted MEF tissues were collected from nude mice euthanized by carbon dioxide inhalation at 6 weeks after MEFs implantation. Representative images show Epo staining (top). Quantification of anti-Epo staining (bottom). n =5 per group; * P <0.01 versus WT. C. Tumor growth rate of AMPKα1-KO MEFs in nude mice injected intraperitoneally (IP) with an antibody specific to Epo or IgG. n =8–10 per group, † P <0.01 versus WT/IgG; * P <0.01 versus α1-KO/IgG. D. Representative images showing staining with antibodies specific to CD-31 or smooth muscle (SM)-α-actin (left). Scale bar =50 μm. Quantification of anti-CD31or SM-α-actin staining (right). Implanted AMPKα1-KO MEFs were collected from nude mice euthanized by carbon dioxide inhalation at the end of experiment (8 weeks after MEFs implantation). n =5–6 per group; * P <0.01 versus IgG. E. Mechanisms of AMPKα1deletion-stimulated anchorage-independent growth, neovascularization, and consequent tumorigenesis. AMPKα1 deletion in fibroblast activates NIK, which phosphorylates and activates IκKα, then enhance p100 phosphorylation recruiting E3 ubiquitin ligase β-TrCP, which facilitates p100 processing to p52. Upregulated p52 controls CDK2 and Epo expression. Epo-mediated neovascularization cooperating with CDK2-mediated anchorage-independent growth contributes to tumorigenesis in vivo .

Journal: Oncotarget

Article Title: AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2

doi: 10.18632/oncotarget.10687

Figure Lengend Snippet: A. Plasma concentrations of Epo in nude mice inoculated with WT or AMPKα1-KO MEFs. n =10-13 per group; * P <0.01 versus WT. B. Epo upregulation in inoculated AMPKα1-KO MEFs in nude mice. Implanted MEF tissues were collected from nude mice euthanized by carbon dioxide inhalation at 6 weeks after MEFs implantation. Representative images show Epo staining (top). Quantification of anti-Epo staining (bottom). n =5 per group; * P <0.01 versus WT. C. Tumor growth rate of AMPKα1-KO MEFs in nude mice injected intraperitoneally (IP) with an antibody specific to Epo or IgG. n =8–10 per group, † P <0.01 versus WT/IgG; * P <0.01 versus α1-KO/IgG. D. Representative images showing staining with antibodies specific to CD-31 or smooth muscle (SM)-α-actin (left). Scale bar =50 μm. Quantification of anti-CD31or SM-α-actin staining (right). Implanted AMPKα1-KO MEFs were collected from nude mice euthanized by carbon dioxide inhalation at the end of experiment (8 weeks after MEFs implantation). n =5–6 per group; * P <0.01 versus IgG. E. Mechanisms of AMPKα1deletion-stimulated anchorage-independent growth, neovascularization, and consequent tumorigenesis. AMPKα1 deletion in fibroblast activates NIK, which phosphorylates and activates IκKα, then enhance p100 phosphorylation recruiting E3 ubiquitin ligase β-TrCP, which facilitates p100 processing to p52. Upregulated p52 controls CDK2 and Epo expression. Epo-mediated neovascularization cooperating with CDK2-mediated anchorage-independent growth contributes to tumorigenesis in vivo .

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): rabbit anti-CDK2 (2546), rabbit anti-pCDK2-T160 (2561), rabbit anti-pp100-S866/870 (4810), rabbit anti-pIĸKα/β (2078), rabbit anti-IĸKα (2682), rabbit anti-NF-ĸB2 p100/p52 (4882), rabbit anti-NIK (4994), rabbit anti-β-TrCP (D13F10) (4394), and rabbit anti-PCNA (13110). β-TrCP siRNA (sc-37179), p52 siRNA (m) (sc-36043), CDK2 shRNA (m) lentiviral particles (sc-29260-V), NFκB p52 shRNA (m) lentiviral particles (sc-36043-V), goat anti-pNIK-T559 (sc-12957), rabbit anti-Epo (sc-7956), mouse anti-GAPDH (sc-32233), mouse anti-β-actin (sc-47778), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Staining, Injection, Phospho-proteomics, Ubiquitin Proteomics, Expressing, In Vivo