Structured Review

Cell Signaling Technology Inc p egfr
Effect of osimertinib on the expression of PIM1, <t>caspase</t> 3, and PARP, and the total levels or phosphorylation inhibition of STAT3, <t>EGFR,</t> AKT, and ERK. H1975 and PC9 cells were treated with vehicle, 5 µM CX-6258 HCl, 5 µM osimertinib, or a combination of both (5 µM) for 6 h following EGF stimulation, and the extracts were blotted using the indicated antibodies. EGFR, epidermal growth factor receptor.
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Images

1) Product Images from "PIM1 inhibitor synergizes the anti-tumor effect of osimertinib via STAT3 dephosphorylation in EGFR-mutant non-small cell lung cancer"

Article Title: PIM1 inhibitor synergizes the anti-tumor effect of osimertinib via STAT3 dephosphorylation in EGFR-mutant non-small cell lung cancer

Journal: Annals of Translational Medicine

doi: 10.21037/atm.2020.02.43

Effect of osimertinib on the expression of PIM1, caspase 3, and PARP, and the total levels or phosphorylation inhibition of STAT3, EGFR, AKT, and ERK. H1975 and PC9 cells were treated with vehicle, 5 µM CX-6258 HCl, 5 µM osimertinib, or a combination of both (5 µM) for 6 h following EGF stimulation, and the extracts were blotted using the indicated antibodies. EGFR, epidermal growth factor receptor.
Figure Legend Snippet: Effect of osimertinib on the expression of PIM1, caspase 3, and PARP, and the total levels or phosphorylation inhibition of STAT3, EGFR, AKT, and ERK. H1975 and PC9 cells were treated with vehicle, 5 µM CX-6258 HCl, 5 µM osimertinib, or a combination of both (5 µM) for 6 h following EGF stimulation, and the extracts were blotted using the indicated antibodies. EGFR, epidermal growth factor receptor.

Techniques Used: Expressing, Inhibition

CX-6258 HCl combined with osimertinib inhibited the expression of PIM1, p-EGFR, and caspase-3 in the tumor tissue. Immunohistochemistry images for PIM1, p-EGFR, and caspase-3 staining of tumor tissue in BALB/c nu/nu mice. All images were 20× magnification; bar =50 µm.
Figure Legend Snippet: CX-6258 HCl combined with osimertinib inhibited the expression of PIM1, p-EGFR, and caspase-3 in the tumor tissue. Immunohistochemistry images for PIM1, p-EGFR, and caspase-3 staining of tumor tissue in BALB/c nu/nu mice. All images were 20× magnification; bar =50 µm.

Techniques Used: Expressing, Immunohistochemistry, Staining, Mouse Assay

2) Product Images from "Nimotuzumab enhances temozolomide‐induced growth suppression of glioma cells expressing mutant EGFR in vivo"

Article Title: Nimotuzumab enhances temozolomide‐induced growth suppression of glioma cells expressing mutant EGFR in vivo

Journal: Cancer Medicine

doi: 10.1002/cam4.614

Dephosphorylation of EGFR v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . Akt phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.
Figure Legend Snippet: Dephosphorylation of EGFR v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . Akt phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.

Techniques Used: De-Phosphorylation Assay, In Vitro, Cell Culture, Western Blot, Expressing, Positive Control, Inhibition

3) Product Images from "CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway"

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4487

CX3CL1/CX3CR1 activates the Src/FAK pathway. (A) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was determined by western blotting. (B) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of EGFP, p-EGFR (Tyr992), PTK2B and p-PTK2B (Tyr402) was measured by western blotting. The expression of EGFR, p-EGFR (Tyr992), Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was measured by western blotting in CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (C) VCaP; (D) PC-3. PC-3 cells were pretreated with (E) bosutinib (0.5 nM for 3 h) or with (F) PF-562271 (0.2 nM for 0.5 h), following which CX3CL1 was added to cells (100 nM for 5, 15, 30, 45 or 60 min), and the expression of p-Src (Tyr416) and p-FAK (Tyr576/577) was examined. VCaP cells were pretreated with either afatinib (1 or 10 µ M for 4 h) or bosutinib (0.25 or 2 µ M for 1 h), following which CX3CL1 (100 nM) was added to cells (100 nM) for 30 min, and the expression of (G) Src, p-Src (Tyr416), (H) EGFR and p-EGFR (Tyr992) were detected by western blotting. (I and J) The expression of MMP-9 and ROCK1 was measured by western blotting CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (I) VCaP; (J) PC-3. Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; EGFR, epidermal growth factor receptor; p, phosphorylated; OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; PTK2B, protein-tyrosine kinase 2-β; CX3CR1, C-X3-C motif chemokine receptor 1; MMP-9, matrix metal-loproteinase-9; ROCK1, Rho-associated protein kinase 1.
Figure Legend Snippet: CX3CL1/CX3CR1 activates the Src/FAK pathway. (A) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was determined by western blotting. (B) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of EGFP, p-EGFR (Tyr992), PTK2B and p-PTK2B (Tyr402) was measured by western blotting. The expression of EGFR, p-EGFR (Tyr992), Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was measured by western blotting in CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (C) VCaP; (D) PC-3. PC-3 cells were pretreated with (E) bosutinib (0.5 nM for 3 h) or with (F) PF-562271 (0.2 nM for 0.5 h), following which CX3CL1 was added to cells (100 nM for 5, 15, 30, 45 or 60 min), and the expression of p-Src (Tyr416) and p-FAK (Tyr576/577) was examined. VCaP cells were pretreated with either afatinib (1 or 10 µ M for 4 h) or bosutinib (0.25 or 2 µ M for 1 h), following which CX3CL1 (100 nM) was added to cells (100 nM) for 30 min, and the expression of (G) Src, p-Src (Tyr416), (H) EGFR and p-EGFR (Tyr992) were detected by western blotting. (I and J) The expression of MMP-9 and ROCK1 was measured by western blotting CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (I) VCaP; (J) PC-3. Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; EGFR, epidermal growth factor receptor; p, phosphorylated; OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; PTK2B, protein-tyrosine kinase 2-β; CX3CR1, C-X3-C motif chemokine receptor 1; MMP-9, matrix metal-loproteinase-9; ROCK1, Rho-associated protein kinase 1.

Techniques Used: Expressing, Western Blot, Over Expression, Negative Control

EGFR, Src and FAK inhibitors reverse cell migration induced by CX3CL1 in PC-3 cells. (A) Cell migration was measured by scratch wound assay. Representative images are presented (×200 magnification). (B) The results were summarized from three independent experiments. * P
Figure Legend Snippet: EGFR, Src and FAK inhibitors reverse cell migration induced by CX3CL1 in PC-3 cells. (A) Cell migration was measured by scratch wound assay. Representative images are presented (×200 magnification). (B) The results were summarized from three independent experiments. * P

Techniques Used: Migration, Scratch Wound Assay Assay

Kinases associated with the CX3CL1/CX3CR1 axis. The association between kinases relevant to CX3CL1 in the Src/focal adhesion kinase signaling pathway were predicted using the Ingenuity Pathway Analysis database. Src, proto-oncogene tyrosine-protein kinase Src; PI3K, phosphatidylinositol 3-kinase; ITGB3, integrin β-3; EGFR, epidermal growth factor receptor; PTK2B, protein-tyrosine kinase 2-β.
Figure Legend Snippet: Kinases associated with the CX3CL1/CX3CR1 axis. The association between kinases relevant to CX3CL1 in the Src/focal adhesion kinase signaling pathway were predicted using the Ingenuity Pathway Analysis database. Src, proto-oncogene tyrosine-protein kinase Src; PI3K, phosphatidylinositol 3-kinase; ITGB3, integrin β-3; EGFR, epidermal growth factor receptor; PTK2B, protein-tyrosine kinase 2-β.

Techniques Used:

4) Product Images from "The prostaglandin receptor EP2 activates multiple signaling pathways and ?-arrestin1 complex formation during mouse skin papilloma development"

Article Title: The prostaglandin receptor EP2 activates multiple signaling pathways and ?-arrestin1 complex formation during mouse skin papilloma development

Journal:

doi: 10.1093/carcin/bgp168

CAY increased cAMP/PKA and Src/EGFR activation in papillomas and surrounding skin. In (B–E), skin or two to three papillomas from three individual mice were combined (six to nine papillomas total) for analysis, and each experiment was repeated.
Figure Legend Snippet: CAY increased cAMP/PKA and Src/EGFR activation in papillomas and surrounding skin. In (B–E), skin or two to three papillomas from three individual mice were combined (six to nine papillomas total) for analysis, and each experiment was repeated.

Techniques Used: Activation Assay, Mouse Assay

Comparison of TPA-induced PKA and EGFR signaling in surrounding skin and papillomas and the effects of their inhibition on papilloma formation. ( A ) Differences in p-CREB (Ser133), EGFR, p-EGFR (Tyr845), H-Ras, p-Src (Tyr416), p-AKT (Ser473) and p-ERK1/2
Figure Legend Snippet: Comparison of TPA-induced PKA and EGFR signaling in surrounding skin and papillomas and the effects of their inhibition on papilloma formation. ( A ) Differences in p-CREB (Ser133), EGFR, p-EGFR (Tyr845), H-Ras, p-Src (Tyr416), p-AKT (Ser473) and p-ERK1/2

Techniques Used: Inhibition

5) Product Images from "PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells, et al. PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells"

Article Title: PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells, et al. PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.14894

PRMT5 activates EGFR/AKT/β‐catenin signalling in pancreatic cancer cells. A, Wnt/β‐catenin and EGFR signalling relative proteins were detected by Western blot in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells. B, After transfected with pHA‐Venus or pHA‐PRMT5 plasmid in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells, EGFR/AKT/β‐catenin signalling relative proteins were detected by Western blot. C, Pancreatic cancer cells were treated with EGFR inhibitor at 0, 10 μmol/L for 3 d. The expression of EGFR, p‐EGFR (Y1068) and PRMT5 was determined by Western blotting. D, The quantification of EGFR and p‐EGFR is shown. ** P
Figure Legend Snippet: PRMT5 activates EGFR/AKT/β‐catenin signalling in pancreatic cancer cells. A, Wnt/β‐catenin and EGFR signalling relative proteins were detected by Western blot in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells. B, After transfected with pHA‐Venus or pHA‐PRMT5 plasmid in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells, EGFR/AKT/β‐catenin signalling relative proteins were detected by Western blot. C, Pancreatic cancer cells were treated with EGFR inhibitor at 0, 10 μmol/L for 3 d. The expression of EGFR, p‐EGFR (Y1068) and PRMT5 was determined by Western blotting. D, The quantification of EGFR and p‐EGFR is shown. ** P

Techniques Used: Western Blot, Infection, Transfection, Plasmid Preparation, Expressing

6) Product Images from "ALK is a therapeutic target for lethal sepsis"

Article Title: ALK is a therapeutic target for lethal sepsis

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aan5689

ALK/EGFR binding triggers AKT-dependent STING activation ( A ) Western blot analysis of indicated protein expression in iBMDMs and RAW264.7 and THP1 cells after stimulation with 3′3′-cGAMP (10 μg/ml), c-di-AMP (10 μg/ml), or DMXAA (10 μg/ml) for 16 hours. ( B ) Heatmap of RTKs phosphorylation changes in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without pharmacologic (LDK378, 10 μM) or genetic inhibition of ALK. ( C ) Relative EGFR phosphorylation assayed in parallel to (B). ( D ) Immunoprecipitation (IP) analysis of the interaction between ALK and EGFR in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without LDK378 (10 μM) or OSI-420 (10 μM). IB, immunoblotting. ( E ) Western blot analysis of indicated protein expression in iBMDMs after treatment with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM). ( F ) Western blot analysis of EGFR expression in EGFR stable knockdown iBMDMs. ( G ) Western blot analysis of indicated protein expression in EGFR-WT and EGFR-knockdown iBMDMs after stimulation with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours. ( H and I ) iBMDMs were treated with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM), and IFNβ protein release (H) and IFNβ mRNA expression (I) were assayed (n = 3; data are means ± SD; * P
Figure Legend Snippet: ALK/EGFR binding triggers AKT-dependent STING activation ( A ) Western blot analysis of indicated protein expression in iBMDMs and RAW264.7 and THP1 cells after stimulation with 3′3′-cGAMP (10 μg/ml), c-di-AMP (10 μg/ml), or DMXAA (10 μg/ml) for 16 hours. ( B ) Heatmap of RTKs phosphorylation changes in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without pharmacologic (LDK378, 10 μM) or genetic inhibition of ALK. ( C ) Relative EGFR phosphorylation assayed in parallel to (B). ( D ) Immunoprecipitation (IP) analysis of the interaction between ALK and EGFR in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without LDK378 (10 μM) or OSI-420 (10 μM). IB, immunoblotting. ( E ) Western blot analysis of indicated protein expression in iBMDMs after treatment with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM). ( F ) Western blot analysis of EGFR expression in EGFR stable knockdown iBMDMs. ( G ) Western blot analysis of indicated protein expression in EGFR-WT and EGFR-knockdown iBMDMs after stimulation with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours. ( H and I ) iBMDMs were treated with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM), and IFNβ protein release (H) and IFNβ mRNA expression (I) were assayed (n = 3; data are means ± SD; * P

Techniques Used: Binding Assay, Activation Assay, Western Blot, Expressing, Inhibition, Immunoprecipitation

7) Product Images from "Delayed administration of suramin attenuates peritoneal fibrosis in rats"

Article Title: Delayed administration of suramin attenuates peritoneal fibrosis in rats

Journal: BMC Nephrology

doi: 10.1186/s12882-019-1597-2

Suramin treatment suppresses the phosphorylation of EGFR, Stat3 and ERK1/2 in peritoneal tissue. Peritoneal lysates were subjected to immunoblot analysis with antibodies to phosphorylated EGFR (p-EGFR), phospho-ERK1/2 (p-ERK1/2), phosphorylated Stat3 (p-STAT3), EGFR, ERK1/2, Stat3, or GAPDH ( a ). Expression levels of p-EGFR were quantified by densitometry and normalized with total EGFR ( b ). Expression levels of p-ERK1/2 were quantified by densitometry and normalized with total ERK1/2 ( c ). Expression levels of p-Stat3 were quantified by densitometry and normalized with total Stat3 ( d ). Data are represented as the mean ± S.E.M. ( n = 6). Means with different lowercase letters are significantly different from one another ( P
Figure Legend Snippet: Suramin treatment suppresses the phosphorylation of EGFR, Stat3 and ERK1/2 in peritoneal tissue. Peritoneal lysates were subjected to immunoblot analysis with antibodies to phosphorylated EGFR (p-EGFR), phospho-ERK1/2 (p-ERK1/2), phosphorylated Stat3 (p-STAT3), EGFR, ERK1/2, Stat3, or GAPDH ( a ). Expression levels of p-EGFR were quantified by densitometry and normalized with total EGFR ( b ). Expression levels of p-ERK1/2 were quantified by densitometry and normalized with total ERK1/2 ( c ). Expression levels of p-Stat3 were quantified by densitometry and normalized with total Stat3 ( d ). Data are represented as the mean ± S.E.M. ( n = 6). Means with different lowercase letters are significantly different from one another ( P

Techniques Used: Expressing

8) Product Images from "EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells"

Article Title: EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells

Journal: Journal of Cancer

doi: 10.7150/jca.44234

EGFR activation is associated with IRE1α -XBP1s Signaling. A. Phosphorylation of EGFR and IRE1α were examined in SW480 cells 30min after stimulation of EGF (50ng/ml). B. Expression of spliced XBP1 mRNA in SW480 cells30 min after stimulation of EGF (50ng/ml). Values are represented as the mean ± SD (n = 3) for each treatment (*P
Figure Legend Snippet: EGFR activation is associated with IRE1α -XBP1s Signaling. A. Phosphorylation of EGFR and IRE1α were examined in SW480 cells 30min after stimulation of EGF (50ng/ml). B. Expression of spliced XBP1 mRNA in SW480 cells30 min after stimulation of EGF (50ng/ml). Values are represented as the mean ± SD (n = 3) for each treatment (*P

Techniques Used: Activation Assay, Expressing

IRE1α-XBP1s pathway and EGFR expression in colorectal cancer. A. Immunoblotting of EGFR, IRE1α and XBP1s in paired tumor and normal tissues of colorectal cancer patients. GAPDH was used as a loading control. B. Expression of spliced XBP1 mRNA in 6 paired patient samples via q-PCR (n=6). Values are represented as the mean ± SD (n = 3) for each treatment (*P
Figure Legend Snippet: IRE1α-XBP1s pathway and EGFR expression in colorectal cancer. A. Immunoblotting of EGFR, IRE1α and XBP1s in paired tumor and normal tissues of colorectal cancer patients. GAPDH was used as a loading control. B. Expression of spliced XBP1 mRNA in 6 paired patient samples via q-PCR (n=6). Values are represented as the mean ± SD (n = 3) for each treatment (*P

Techniques Used: Expressing, Polymerase Chain Reaction

EGFR signaling activates IRE1α through the kinase activity of ERK. A. The molecules of EGFR pathway were detected in SW480 cells by immunoblotting. B. The molecules of EGFR pathway of HCT116 EGFRKO cell line and HCT116 cells were examined by immunoblotting. C. EGFR downstream pathway were detected by immunoblotting in HCT116 treated with cetuximab (12.5μg/ml) for 24hrs. D. Proteins of IRE1α-XBP1s pathway in HCT116 cells with the treatment of MEK inhibitor PD0325901 (1nM) for 24hrs were assessed by immunoblotting. E. p-IRE1α (S724) and XBP1s were assessed via immunoblotting after the treatment of MEK inhibitor PD0325901 (1nM) or ERK inhibitor SCH8477 (5μM) for 24hrs. F. Expression of spliced XBP1 mRNA was assessed in SW480 cells after treatment with MEK inhibitor PD0325901 (1nM) or ERK inhibitor SCH8477 (5μM). MKC8866 was used as positive control. Values are represented as the mean ± SD (n = 3) for each treatment (****P
Figure Legend Snippet: EGFR signaling activates IRE1α through the kinase activity of ERK. A. The molecules of EGFR pathway were detected in SW480 cells by immunoblotting. B. The molecules of EGFR pathway of HCT116 EGFRKO cell line and HCT116 cells were examined by immunoblotting. C. EGFR downstream pathway were detected by immunoblotting in HCT116 treated with cetuximab (12.5μg/ml) for 24hrs. D. Proteins of IRE1α-XBP1s pathway in HCT116 cells with the treatment of MEK inhibitor PD0325901 (1nM) for 24hrs were assessed by immunoblotting. E. p-IRE1α (S724) and XBP1s were assessed via immunoblotting after the treatment of MEK inhibitor PD0325901 (1nM) or ERK inhibitor SCH8477 (5μM) for 24hrs. F. Expression of spliced XBP1 mRNA was assessed in SW480 cells after treatment with MEK inhibitor PD0325901 (1nM) or ERK inhibitor SCH8477 (5μM). MKC8866 was used as positive control. Values are represented as the mean ± SD (n = 3) for each treatment (****P

Techniques Used: Activity Assay, Expressing, Positive Control

9) Product Images from "HIF-1α links β-adrenoceptor agonists and pancreatic cancer cells under normoxic condition"

Article Title: HIF-1α links β-adrenoceptor agonists and pancreatic cancer cells under normoxic condition

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2009.181

Phosphorylation of EGFR (Tyr 1173, Tyr1608, Tyr992) in response to β-AR agonists and antagonists. (A) MIA PaCa2 and BxPC-3 cells were treated with drugs as indicated below the panel and tyrosine phosphorylation at 3 sites within EGFR was assessed using specific antibodies. EGF provided the positive control. (B) Quantitation of Western blotting. Data from at least 3 independent experiments expressed as means±SEM vs controls. b P
Figure Legend Snippet: Phosphorylation of EGFR (Tyr 1173, Tyr1608, Tyr992) in response to β-AR agonists and antagonists. (A) MIA PaCa2 and BxPC-3 cells were treated with drugs as indicated below the panel and tyrosine phosphorylation at 3 sites within EGFR was assessed using specific antibodies. EGF provided the positive control. (B) Quantitation of Western blotting. Data from at least 3 independent experiments expressed as means±SEM vs controls. b P

Techniques Used: Positive Control, Quantitation Assay, Western Blot

10) Product Images from "Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells"

Article Title: Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells

Journal: Scientific Reports

doi: 10.1038/srep18430

β1 regulated cell signaling with a cell density-dependent manner and was dispensable for EGFR dimer formation. ( A ) Low, middle and high densities of cell lysates were immunoblotted by anti-pEGFR, anti-EGFR, anti-pSrc, anti-Src, anti-pERK, anti-ERK, anti-pAKT and anti-AKT antibodies. α-Tubulin was used as a loading control. ( B ) Graphical representation of relative level of pEGFR, pSrc, pERK and pAKT in low, middle and high densities of MDA-MB-231 cells, respectively. Data are represented as the means ± s.e.m of three independent experiments (* p
Figure Legend Snippet: β1 regulated cell signaling with a cell density-dependent manner and was dispensable for EGFR dimer formation. ( A ) Low, middle and high densities of cell lysates were immunoblotted by anti-pEGFR, anti-EGFR, anti-pSrc, anti-Src, anti-pERK, anti-ERK, anti-pAKT and anti-AKT antibodies. α-Tubulin was used as a loading control. ( B ) Graphical representation of relative level of pEGFR, pSrc, pERK and pAKT in low, middle and high densities of MDA-MB-231 cells, respectively. Data are represented as the means ± s.e.m of three independent experiments (* p

Techniques Used: Multiple Displacement Amplification

11) Product Images from "A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma"

Article Title: A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma

Journal: Cancer Cell International

doi: 10.1186/s12935-017-0496-5

Notch1 C1133Y mutation activates the EGFR-PI3K/AKT signaling pathway. a The efficiency of Notch1 transfection was verified in HN6, CAL27 and HN13 cells. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1 C1133Y mutation reversed the trend in all the tested cell lines. Notch1 C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection. b The grey-scale analysis of the alteration of EGFR pathway in panel A
Figure Legend Snippet: Notch1 C1133Y mutation activates the EGFR-PI3K/AKT signaling pathway. a The efficiency of Notch1 transfection was verified in HN6, CAL27 and HN13 cells. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1 C1133Y mutation reversed the trend in all the tested cell lines. Notch1 C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection. b The grey-scale analysis of the alteration of EGFR pathway in panel A

Techniques Used: Mutagenesis, Transfection, Western Blot, Expressing

Schematic model for EGFR-PI3K/AKT signaling activation induced by Notch1 C1133Y mutation. a Schematic depiction of Abruptex domain C1133Y mutation and S1-3 cleavages in Notch1 receptor protein. EGF epidermal growth factor, LNR Lin/Notch repeats, HD (N and C regions), heterodimerization domain, TM transmembrane domain, RAM RBP-Jκ-associated molecule region, ANK ankyrin repeats, TAD transactivation domain, PEST sequence rich in proline, glutamic acid, serine, and threonine. S1-3, S1-3 cleavages. Black arrows indicate the sites of the cleavages. Red arrow indicates the site of the C1133Y mutation. b Model for aberrant EGFR-PI3K/AKT signaling pathway activation by Notch1 C1133Y mutation. The Notch1 protein is synthesized in endoplasmic reticulum and is transported to Golgi complex for S1-cleavage. The S1-cleaved mature Notch1 protein is presented on cell surface, where it has an inhibitory effect on EGFR phosphorylation. The ligand binding causes cleavage of the receptor at the S2-cleavage site. The remaining Notch1 receptor undergoes further cleavage at the S3 site, freeing the NICD domain. The NICD translocates to the nucleus where it binds to the DNA-binding protein CSL and was recognized by the transcriptional coactivator Mastermind (MAM). The triprotein complex recruits additional coactivators (Co-A) to activate target genes. In this study, we find that the Notch1 signaling has an inhibitory effect on EGFR activation. When Notch1 C1133Y mutation occurs, Notch1 protein is arrested in endoplasmic reticulum and is unable to be transported to Golgi complex for S1-cleavage, thus the canonical Notch1 signaling activation is disrupted. The PI3K/AKT signaling is activated by Notch1 protein arrest in endoplasmic reticulum induced by Notch1 C1133Y mutation. Moreover, the loss of inhibitory effect by Notch1 loss-of-function mutation can also induces EGFR phosphorylation, thus activating PI3K/AKT signaling. NECD Notch1 extracellular domain, NICD Notch1 intracellular domain
Figure Legend Snippet: Schematic model for EGFR-PI3K/AKT signaling activation induced by Notch1 C1133Y mutation. a Schematic depiction of Abruptex domain C1133Y mutation and S1-3 cleavages in Notch1 receptor protein. EGF epidermal growth factor, LNR Lin/Notch repeats, HD (N and C regions), heterodimerization domain, TM transmembrane domain, RAM RBP-Jκ-associated molecule region, ANK ankyrin repeats, TAD transactivation domain, PEST sequence rich in proline, glutamic acid, serine, and threonine. S1-3, S1-3 cleavages. Black arrows indicate the sites of the cleavages. Red arrow indicates the site of the C1133Y mutation. b Model for aberrant EGFR-PI3K/AKT signaling pathway activation by Notch1 C1133Y mutation. The Notch1 protein is synthesized in endoplasmic reticulum and is transported to Golgi complex for S1-cleavage. The S1-cleaved mature Notch1 protein is presented on cell surface, where it has an inhibitory effect on EGFR phosphorylation. The ligand binding causes cleavage of the receptor at the S2-cleavage site. The remaining Notch1 receptor undergoes further cleavage at the S3 site, freeing the NICD domain. The NICD translocates to the nucleus where it binds to the DNA-binding protein CSL and was recognized by the transcriptional coactivator Mastermind (MAM). The triprotein complex recruits additional coactivators (Co-A) to activate target genes. In this study, we find that the Notch1 signaling has an inhibitory effect on EGFR activation. When Notch1 C1133Y mutation occurs, Notch1 protein is arrested in endoplasmic reticulum and is unable to be transported to Golgi complex for S1-cleavage, thus the canonical Notch1 signaling activation is disrupted. The PI3K/AKT signaling is activated by Notch1 protein arrest in endoplasmic reticulum induced by Notch1 C1133Y mutation. Moreover, the loss of inhibitory effect by Notch1 loss-of-function mutation can also induces EGFR phosphorylation, thus activating PI3K/AKT signaling. NECD Notch1 extracellular domain, NICD Notch1 intracellular domain

Techniques Used: Activation Assay, Mutagenesis, Sequencing, Synthesized, Ligand Binding Assay, Binding Assay

Notch1 C1133Y mutation accelerates cell proliferation. a – c The CCK-8 growth curves of HN6, CAL27 and HN13 cell lines after transfections. The overexpression of wild-type Notch1 attenuated cell growth in HN6, but it enhanced cell growth in CAL27 and HN13, compared with controls. However, Notch1 C1133Y mutation accelerated cell growth in all the tested cell lines, compared with Notch1 WT -transfected cells. d The cell-cycle analysis by flow cytometry revealed a less G1 phase arrest in Notch1 C1133Y -transfected cells. e Cell-cycle-specific proteins were analyzed by western blot analysis in HN6 and HN13. Notch1 C1133Y transfection resulted in the up-regulation of CDKs (2 and 4) and cyclins (D1 and D3), whereas the expression of P27 and P21 was decreased, suggesting an expedited cell-cycle induced by Notch1 C1133Y transfection. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1 C1133Y mutation reversed the trend in all the tested cell lines. Notch1 C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection
Figure Legend Snippet: Notch1 C1133Y mutation accelerates cell proliferation. a – c The CCK-8 growth curves of HN6, CAL27 and HN13 cell lines after transfections. The overexpression of wild-type Notch1 attenuated cell growth in HN6, but it enhanced cell growth in CAL27 and HN13, compared with controls. However, Notch1 C1133Y mutation accelerated cell growth in all the tested cell lines, compared with Notch1 WT -transfected cells. d The cell-cycle analysis by flow cytometry revealed a less G1 phase arrest in Notch1 C1133Y -transfected cells. e Cell-cycle-specific proteins were analyzed by western blot analysis in HN6 and HN13. Notch1 C1133Y transfection resulted in the up-regulation of CDKs (2 and 4) and cyclins (D1 and D3), whereas the expression of P27 and P21 was decreased, suggesting an expedited cell-cycle induced by Notch1 C1133Y transfection. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1 C1133Y mutation reversed the trend in all the tested cell lines. Notch1 C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection

Techniques Used: Mutagenesis, CCK-8 Assay, Transfection, Over Expression, Cell Cycle Assay, Flow Cytometry, Cytometry, Western Blot, Expressing

12) Product Images from "Boehmenan, a Lignan From the Chinese Medicinal Plant Clematis armandii, Inhibits A431 Cell Growth via Blocking p70S6/S6 Kinase Pathway"

Article Title: Boehmenan, a Lignan From the Chinese Medicinal Plant Clematis armandii, Inhibits A431 Cell Growth via Blocking p70S6/S6 Kinase Pathway

Journal: Integrative Cancer Therapies

doi: 10.1177/1534735416669803

Boehmenan inhibited EGF-induced STAT3 activation. A431 cells were pretreated with boehmenan (6.25-25 µM) for 4 hours and then stimulated with EGF (100 ng/mL) for 30 minutes; the expression of phosphor (p)- and total-EGFR, MEK, ERK, and STAT3 was analyzed by Western blot. Bar graphs and quantitative analysis for p-EGFR/EGFR (A), p-STAT3/STAT3 (B), p-MEK/MEK (C), and p-ERK/ERK (D). Data shown are means ± SEM. $ P
Figure Legend Snippet: Boehmenan inhibited EGF-induced STAT3 activation. A431 cells were pretreated with boehmenan (6.25-25 µM) for 4 hours and then stimulated with EGF (100 ng/mL) for 30 minutes; the expression of phosphor (p)- and total-EGFR, MEK, ERK, and STAT3 was analyzed by Western blot. Bar graphs and quantitative analysis for p-EGFR/EGFR (A), p-STAT3/STAT3 (B), p-MEK/MEK (C), and p-ERK/ERK (D). Data shown are means ± SEM. $ P

Techniques Used: Activation Assay, Expressing, Western Blot

13) Product Images from "The expression of keratin 6 is regulated by the activation of the ERK1/2 pathway in arsenite transformed human urothelial cells"

Article Title: The expression of keratin 6 is regulated by the activation of the ERK1/2 pathway in arsenite transformed human urothelial cells

Journal: Toxicology and applied pharmacology

doi: 10.1016/j.taap.2017.05.007

Effect of EGF on the expression of KRT6 and the activation of EGFR and downstream kinases in the UROtsa parent cells. (A and B). Western blot analysis of KRT6 expression in UROtsa parent cells after treatment with EGF for various time periods. The integrated optical densities (IOD) for each of the KRT6 band/β-actin is indicated. (A – F). Phosphorylation of EGFR (A and C), ERK1/2 (A and D), JNK (A and E) and AKT (A and F) was determined by Western blotting. The IOD for each phosphorylated protein is plotted per total protein. * indicates significantly different at p
Figure Legend Snippet: Effect of EGF on the expression of KRT6 and the activation of EGFR and downstream kinases in the UROtsa parent cells. (A and B). Western blot analysis of KRT6 expression in UROtsa parent cells after treatment with EGF for various time periods. The integrated optical densities (IOD) for each of the KRT6 band/β-actin is indicated. (A – F). Phosphorylation of EGFR (A and C), ERK1/2 (A and D), JNK (A and E) and AKT (A and F) was determined by Western blotting. The IOD for each phosphorylated protein is plotted per total protein. * indicates significantly different at p

Techniques Used: Expressing, Activation Assay, Western Blot

14) Product Images from "Intranasal delivery of VEGF enhances compensatory lung growth in mice"

Article Title: Intranasal delivery of VEGF enhances compensatory lung growth in mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0198700

Lung tissue protein expression analyses. ELISA reveals increased VEGF levels in VEGF-treated lungs (A) on POD 4. However, quantitative polymerase chain reactions (qPCR) show no difference in mRNA transcript levels of VEGF, VEGFR1, or VEGFR2 between the two groups (B). Immunoblot demonstrates an increase in the levels of P-VEGFR2, VEGFR2, P-EGFR, EGFR, and heparin-binding EGF-like growth factor (HB-EGF) with VEGF treatment (C-D). Data are expressed as mean ± SE.
Figure Legend Snippet: Lung tissue protein expression analyses. ELISA reveals increased VEGF levels in VEGF-treated lungs (A) on POD 4. However, quantitative polymerase chain reactions (qPCR) show no difference in mRNA transcript levels of VEGF, VEGFR1, or VEGFR2 between the two groups (B). Immunoblot demonstrates an increase in the levels of P-VEGFR2, VEGFR2, P-EGFR, EGFR, and heparin-binding EGF-like growth factor (HB-EGF) with VEGF treatment (C-D). Data are expressed as mean ± SE.

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Binding Assay

15) Product Images from "Radiosensitivity enhancement by combined treatment of nimotuzumab and celecoxib on nasopharyngeal carcinoma cells"

Article Title: Radiosensitivity enhancement by combined treatment of nimotuzumab and celecoxib on nasopharyngeal carcinoma cells

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S163595

Combined nimotuzumab and celecoxib cooperatively blocks radiation-induced EGFR activation and internalization in CNE2 cell line. CNE2 cells were preincubated with N50 or C25 or the combination for 24 hours, and then exposed to 4 Gy of X-ray radiation and further incubated for 2 hours. Cytoplasmic and nuclear extracts were harvested for Western blot analysis. ( A ) Cytoplasmic levels of EGFR, p-EGFR, p-ERK1/2, p-STAT3, p-AKT and COX-2. ( B ) Nuclear levels of EGFR and p-DNA-PK. Abbreviation: IR, irradiation.
Figure Legend Snippet: Combined nimotuzumab and celecoxib cooperatively blocks radiation-induced EGFR activation and internalization in CNE2 cell line. CNE2 cells were preincubated with N50 or C25 or the combination for 24 hours, and then exposed to 4 Gy of X-ray radiation and further incubated for 2 hours. Cytoplasmic and nuclear extracts were harvested for Western blot analysis. ( A ) Cytoplasmic levels of EGFR, p-EGFR, p-ERK1/2, p-STAT3, p-AKT and COX-2. ( B ) Nuclear levels of EGFR and p-DNA-PK. Abbreviation: IR, irradiation.

Techniques Used: Activation Assay, Incubation, Western Blot, Irradiation

Combined nimotuzumab and celecoxib cooperatively inhibits cytoplasmic and nuclear EGFR signaling pathways in CNE2 cell line. CNE2 cells were incubated with N50 or C25 or the combination for 48 hours. Cytoplasmic and nuclear extracts were harvested for Western blot analysis. ( A ) Cytoplasmic levels of EGFR, p-EGFR, p-ERK1/2, p-STAT3, p-AKT and COX-2. ( B ) Nuclear levels of EGFR and PCNA.
Figure Legend Snippet: Combined nimotuzumab and celecoxib cooperatively inhibits cytoplasmic and nuclear EGFR signaling pathways in CNE2 cell line. CNE2 cells were incubated with N50 or C25 or the combination for 48 hours. Cytoplasmic and nuclear extracts were harvested for Western blot analysis. ( A ) Cytoplasmic levels of EGFR, p-EGFR, p-ERK1/2, p-STAT3, p-AKT and COX-2. ( B ) Nuclear levels of EGFR and PCNA.

Techniques Used: Incubation, Western Blot

16) Product Images from "Gastrokine 1 inhibits gastrin-induced cell proliferation"

Article Title: Gastrokine 1 inhibits gastrin-induced cell proliferation

Journal: Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association

doi: 10.1007/s10120-015-0483-2

GKN1 downregulates gastrin-induced expression of CCKBR, growth factor receptors, and NF-κB-related proteins. a, b Gastrin increased the expression of CCKBR, EGFR, p-c-Met, and c-Met in AGS and MKN1 cells, whereas GKN1 significantly decreased the expression levels of these proteins, even in the cells treated with gastrin. c GKN1 silencing in HFE-145 cells resulted in elevated expression of the CCKBR, EGFR, and c-Met proteins. d Gastrin enhanced the expression of p-p65 and slightly increased that of IKKα/β, although it did not affect the expression of p65 and IκB proteins. However, GKN1 reverted the expression of IκB and decreased the expression of IKKα/β, p-p65, and p65 proteins. Data shown are representative of at least three independent experiments. e A significant decline in mRNA expression of gastrin was observed in AGS and MKN1 cells stably expressing the GKN1 protein ( P = 0.015 and P = 0.026, respectively). f Gastrin markedly enhanced CCKBR mRNA expression in AGS and MKN1 cells ( P = 0.017 and P = 0.047, respectively), whereas GKN1 completely suppressed the increase in the CCKBR mRNA levels induced by gastrin in both cell lines ( P = 0.028 and P = 0.024, respectively). Experiments were performed in triplicate. Data are reported as relative quantities, according to an internal calibrator using the 2 −ΔΔCT ]. A Student's t test ( P
Figure Legend Snippet: GKN1 downregulates gastrin-induced expression of CCKBR, growth factor receptors, and NF-κB-related proteins. a, b Gastrin increased the expression of CCKBR, EGFR, p-c-Met, and c-Met in AGS and MKN1 cells, whereas GKN1 significantly decreased the expression levels of these proteins, even in the cells treated with gastrin. c GKN1 silencing in HFE-145 cells resulted in elevated expression of the CCKBR, EGFR, and c-Met proteins. d Gastrin enhanced the expression of p-p65 and slightly increased that of IKKα/β, although it did not affect the expression of p65 and IκB proteins. However, GKN1 reverted the expression of IκB and decreased the expression of IKKα/β, p-p65, and p65 proteins. Data shown are representative of at least three independent experiments. e A significant decline in mRNA expression of gastrin was observed in AGS and MKN1 cells stably expressing the GKN1 protein ( P = 0.015 and P = 0.026, respectively). f Gastrin markedly enhanced CCKBR mRNA expression in AGS and MKN1 cells ( P = 0.017 and P = 0.047, respectively), whereas GKN1 completely suppressed the increase in the CCKBR mRNA levels induced by gastrin in both cell lines ( P = 0.028 and P = 0.024, respectively). Experiments were performed in triplicate. Data are reported as relative quantities, according to an internal calibrator using the 2 −ΔΔCT ]. A Student's t test ( P

Techniques Used: Expressing, Stable Transfection

GKN1 inhibits the c-myc-induced expression of CCKBR. a The ectopic expression of c-myc markedly increased the expression of CCKBR and p-EGFR in AGS and MKN1 cells, whereas GKN1 counteracted the effects of c-myc in both cell lines. Data shown are representative of at least three independent experiments. b, c mRNA levels of gastin and CCKBR were dramatically augmented in c-myc -transfected AGS and MKN1 cells ( P = 0.029 and 0.045, respectively). However, GKN1 significantly downregulated gastrin and CCKBR mRNA expression ( P = 0.008 and 0.01) and abrogated the stimulating effect of c-myc on both genes ( P = 0.02 and 0.031, respectively). Experiments were performed in triplicate. Data are reported as relative quantities, according to an internal calibrator using the 2 −ΔΔCT ]. Unpaired Student's t test ( P
Figure Legend Snippet: GKN1 inhibits the c-myc-induced expression of CCKBR. a The ectopic expression of c-myc markedly increased the expression of CCKBR and p-EGFR in AGS and MKN1 cells, whereas GKN1 counteracted the effects of c-myc in both cell lines. Data shown are representative of at least three independent experiments. b, c mRNA levels of gastin and CCKBR were dramatically augmented in c-myc -transfected AGS and MKN1 cells ( P = 0.029 and 0.045, respectively). However, GKN1 significantly downregulated gastrin and CCKBR mRNA expression ( P = 0.008 and 0.01) and abrogated the stimulating effect of c-myc on both genes ( P = 0.02 and 0.031, respectively). Experiments were performed in triplicate. Data are reported as relative quantities, according to an internal calibrator using the 2 −ΔΔCT ]. Unpaired Student's t test ( P

Techniques Used: Expressing, Transfection

17) Product Images from "Concurrent HER or PI3K Inhibition Potentiates the Anti-tumor Effect of ERK Inhibitor Ulixertinib in Preclinical Pancreatic Cancer Models"

Article Title: Concurrent HER or PI3K Inhibition Potentiates the Anti-tumor Effect of ERK Inhibitor Ulixertinib in Preclinical Pancreatic Cancer Models

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-17-1142

Pan-HER inhibition potentiates the anti-tumor effect of ulixertinib. A, Hypothesized adaptive mechanisms to ulixertinib and proposed combinatorial strategies with afatinib or GDC-0941. B, Western blots showing changes in phospho-EGFR, phospho-HER2 and phospho-HER3 levels in various PDAC lines after overnight treatment with ulixertinib. C, Western blots showing the overnight treatment effect of ulixertinib, afatinib or both on the indicated proteins across the four PDAC lines. D, Relative quantification of soft agar colonies formed by PDAC lines treated as indicated. One of three sets of experiments, each done in triplicates, was presented. E, Median effect analyses of ulixertinib plus afatinib in four indicated PDAC lines as represented by combination indices at six different fixed-ratio concentrations (all in μM) of ulixertinib: afatinib (10:5, 5:2.5, 2.5:1.25, 1.25:0.625; 0.625:0.31, 0.31:0.15). One of three sets of experiments, each done in triplicates, was presented. F, Caspase 3/7 reporter assay showing the pro-apoptotic effect of ulixertinib and/or afatinib in MIA Paca-2 cells. G, Serial measurements of relative tumor volume (to initial) and H, final weight of MIA Paca-2 tumors grown subcutaneously in nude mice treated as indicated when tumors reached ~100mm 3 (N=10/group). I, Serial measurements of relative tumor volume (to initial) and J, final weight of HPNE- KRAS G12D tumors grown subcutaneously in nude mice treated as indicated when tumors reached ~100mm 3 (N=8/group). K, H E (upper panels), p-ERK and p-AKT IHC (middle and lower panels) staining showing the effect of drug treatments on MIA Paca-2 tumors. (*p
Figure Legend Snippet: Pan-HER inhibition potentiates the anti-tumor effect of ulixertinib. A, Hypothesized adaptive mechanisms to ulixertinib and proposed combinatorial strategies with afatinib or GDC-0941. B, Western blots showing changes in phospho-EGFR, phospho-HER2 and phospho-HER3 levels in various PDAC lines after overnight treatment with ulixertinib. C, Western blots showing the overnight treatment effect of ulixertinib, afatinib or both on the indicated proteins across the four PDAC lines. D, Relative quantification of soft agar colonies formed by PDAC lines treated as indicated. One of three sets of experiments, each done in triplicates, was presented. E, Median effect analyses of ulixertinib plus afatinib in four indicated PDAC lines as represented by combination indices at six different fixed-ratio concentrations (all in μM) of ulixertinib: afatinib (10:5, 5:2.5, 2.5:1.25, 1.25:0.625; 0.625:0.31, 0.31:0.15). One of three sets of experiments, each done in triplicates, was presented. F, Caspase 3/7 reporter assay showing the pro-apoptotic effect of ulixertinib and/or afatinib in MIA Paca-2 cells. G, Serial measurements of relative tumor volume (to initial) and H, final weight of MIA Paca-2 tumors grown subcutaneously in nude mice treated as indicated when tumors reached ~100mm 3 (N=10/group). I, Serial measurements of relative tumor volume (to initial) and J, final weight of HPNE- KRAS G12D tumors grown subcutaneously in nude mice treated as indicated when tumors reached ~100mm 3 (N=8/group). K, H E (upper panels), p-ERK and p-AKT IHC (middle and lower panels) staining showing the effect of drug treatments on MIA Paca-2 tumors. (*p

Techniques Used: Inhibition, Western Blot, Reporter Assay, Mouse Assay, Immunohistochemistry, Staining

18) Product Images from "HCRP-1 regulates EGFR–AKT–BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer"

Article Title: HCRP-1 regulates EGFR–AKT–BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1217-2

Hypothetic model of HCRP-1 depletion inhibits BIM expression via the EGFR–AKT signaling pathway
Figure Legend Snippet: Hypothetic model of HCRP-1 depletion inhibits BIM expression via the EGFR–AKT signaling pathway

Techniques Used: Expressing

Suppression of EGFR blocks the loss of HCRP-1 induced by the AKT signaling pathway, upregulates BIM expression, and suppresses anoikis resistance. a , b HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low-attachment surface. The whole-cell lysates were analyzed for the protein levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin by using western blot. c , d Cell anoikis was detected by flow cytometry. HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low- attachment surface. Cells that did not adhere were subjected to flow cytometry detection. e and f HCT116 and SW620 cells were co-transfected with HCRP-1 siRNA and/or EGFR siRNA. Levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin in cells that did not adhere were determined by Western blot. g , h Cell anoikis was detected by flow cytometry in colon cancer cells pretreated with HCRP-1 siRNA and/or EGFR siRNA. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P
Figure Legend Snippet: Suppression of EGFR blocks the loss of HCRP-1 induced by the AKT signaling pathway, upregulates BIM expression, and suppresses anoikis resistance. a , b HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low-attachment surface. The whole-cell lysates were analyzed for the protein levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin by using western blot. c , d Cell anoikis was detected by flow cytometry. HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low- attachment surface. Cells that did not adhere were subjected to flow cytometry detection. e and f HCT116 and SW620 cells were co-transfected with HCRP-1 siRNA and/or EGFR siRNA. Levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin in cells that did not adhere were determined by Western blot. g , h Cell anoikis was detected by flow cytometry in colon cancer cells pretreated with HCRP-1 siRNA and/or EGFR siRNA. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P

Techniques Used: Expressing, Western Blot, Flow Cytometry, Cytometry, Transfection

Inhibition of HCRP-1 activates the EGFR/AKT signaling pathway and suppresses BIM protein expression. a , b HCT116 and SW620 cells were transfected with si-HCRP-1 or control siRNA, and then cells were harvested and submitted to Western blot detection for the protein expression of BIM, FoxO3a, EGFR, AKT, ERK, Mcl-1, Bcl-2, and β-actin. Phosphorylated forms of EGFR, AKT, FoxO3a, and ERK were also detected by western blot with the corresponding antibodies. c–f Cells were treatedwith CHX for 0, 0.5 , 1, and 2 h after transfection with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to western blot detection for the protein expression of EGFR. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P
Figure Legend Snippet: Inhibition of HCRP-1 activates the EGFR/AKT signaling pathway and suppresses BIM protein expression. a , b HCT116 and SW620 cells were transfected with si-HCRP-1 or control siRNA, and then cells were harvested and submitted to Western blot detection for the protein expression of BIM, FoxO3a, EGFR, AKT, ERK, Mcl-1, Bcl-2, and β-actin. Phosphorylated forms of EGFR, AKT, FoxO3a, and ERK were also detected by western blot with the corresponding antibodies. c–f Cells were treatedwith CHX for 0, 0.5 , 1, and 2 h after transfection with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to western blot detection for the protein expression of EGFR. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P

Techniques Used: Inhibition, Expressing, Transfection, Western Blot

19) Product Images from "Upregulation of SALL4 by EGFR activation regulates the stemness of CD44-positive lung cancer"

Article Title: Upregulation of SALL4 by EGFR activation regulates the stemness of CD44-positive lung cancer

Journal: Oncogenesis

doi: 10.1038/s41389-018-0045-7

SALL4 knockdown results into alterations in drug sensitivity of NSCLCs response to EGFR-TKIs. a PC9 cells infected with Lenti-control or SALL4 shRNA were cultivated in medium with and without Erlotinib for 72 h, then the cell viability was assessed. b Cells were kept in medium with and without Erlotinib (100 nM) for 48 h, and the apoptotic cells was measured by staining with Annexin V-PI by FACS. Data are representative of three independent experiments. c Nude mice with xenografts established by subcutaneous injection of PC9 cells infected with Lenti-control or SALL4 shRNA were treated with vehicle or Erlotinib (10 mg/kg). Data are means ± s.d. for 5 mice per group. d Representative imaging of mice injected PC-9 cells treated with or without Erlotinib
Figure Legend Snippet: SALL4 knockdown results into alterations in drug sensitivity of NSCLCs response to EGFR-TKIs. a PC9 cells infected with Lenti-control or SALL4 shRNA were cultivated in medium with and without Erlotinib for 72 h, then the cell viability was assessed. b Cells were kept in medium with and without Erlotinib (100 nM) for 48 h, and the apoptotic cells was measured by staining with Annexin V-PI by FACS. Data are representative of three independent experiments. c Nude mice with xenografts established by subcutaneous injection of PC9 cells infected with Lenti-control or SALL4 shRNA were treated with vehicle or Erlotinib (10 mg/kg). Data are means ± s.d. for 5 mice per group. d Representative imaging of mice injected PC-9 cells treated with or without Erlotinib

Techniques Used: Infection, shRNA, Staining, FACS, Mouse Assay, Injection, Imaging

Association of high SALL4 expression with activated EGFR mutations in lung cancer tissues. a Microarray analysis of SALL4 expression in normal ( n = 20) and lung tumor ( n = 226). b The highest expression of SALL4 in EGFR mutation in comparison with KRAS mutation, ALK-rearrangement, and normal WT tissues. The average value of SALL4 expression is shown in the graph. c SALL4 upregulation is specifically induced by EGFR mutation, but not EGFR WT, KRAS mutation and ALK rearrangement in human lung cancer. d, e Kaplan–Meier survival analysis with Log-rank from the PrognoScan database. Recurrence-free survival and overall survival of SALL4-low and SALL4-high NSCLCs was analyzed. The P -value for the difference between the two curves were determined by the log-rank test
Figure Legend Snippet: Association of high SALL4 expression with activated EGFR mutations in lung cancer tissues. a Microarray analysis of SALL4 expression in normal ( n = 20) and lung tumor ( n = 226). b The highest expression of SALL4 in EGFR mutation in comparison with KRAS mutation, ALK-rearrangement, and normal WT tissues. The average value of SALL4 expression is shown in the graph. c SALL4 upregulation is specifically induced by EGFR mutation, but not EGFR WT, KRAS mutation and ALK rearrangement in human lung cancer. d, e Kaplan–Meier survival analysis with Log-rank from the PrognoScan database. Recurrence-free survival and overall survival of SALL4-low and SALL4-high NSCLCs was analyzed. The P -value for the difference between the two curves were determined by the log-rank test

Techniques Used: Expressing, Microarray, Mutagenesis

EGFR signaling activated by the ligand EGF stimulation and EGFR L858R point mutation induced the expression of SALL4 through ERK1/2 pathway. a qRT-PCR analysis of SALL4 expression in Beas-2B cells treated with different dose of EGF (0, 10, 20, and 40 ng/ml) for 30 min and the protein level of SALL4 was detected by Western blotting. b qRT-PCR and western blotting analysis of SALL4 expression in Bease-2B cells infected with Lenti-control and -EGFR L858R. c qRT-PCR and Western blot analysis of SALL4 expression in PC9 treated with different doses of Gefitinib (0, 5, 10, 100 nM) for 48 h respectively. d The protein level of SALL4, p-EGFR, p-ERK, ERK, p-AKT, and AKT in PC9 cells treated with Gefitinib (100, 500 nM) for 48 h, and β-actin was selected as a control. e The mRNA and protein level of SALL4 in PC9 cells, which were treated with 1 μM ERK inhibitor (SCH772984) and 2 μM AKT inhibitor (MK-22062HCL) for 72 h. Representative results from three independent experiments are shown (mean ± s.d.)
Figure Legend Snippet: EGFR signaling activated by the ligand EGF stimulation and EGFR L858R point mutation induced the expression of SALL4 through ERK1/2 pathway. a qRT-PCR analysis of SALL4 expression in Beas-2B cells treated with different dose of EGF (0, 10, 20, and 40 ng/ml) for 30 min and the protein level of SALL4 was detected by Western blotting. b qRT-PCR and western blotting analysis of SALL4 expression in Bease-2B cells infected with Lenti-control and -EGFR L858R. c qRT-PCR and Western blot analysis of SALL4 expression in PC9 treated with different doses of Gefitinib (0, 5, 10, 100 nM) for 48 h respectively. d The protein level of SALL4, p-EGFR, p-ERK, ERK, p-AKT, and AKT in PC9 cells treated with Gefitinib (100, 500 nM) for 48 h, and β-actin was selected as a control. e The mRNA and protein level of SALL4 in PC9 cells, which were treated with 1 μM ERK inhibitor (SCH772984) and 2 μM AKT inhibitor (MK-22062HCL) for 72 h. Representative results from three independent experiments are shown (mean ± s.d.)

Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR, Western Blot, Infection

20) Product Images from "Downregulation of BANCR Promotes Aggressiveness in Papillary Thyroid Cancer via the MAPK and PI3K Pathways"

Article Title: Downregulation of BANCR Promotes Aggressiveness in Papillary Thyroid Cancer via the MAPK and PI3K Pathways

Journal: Journal of Cancer

doi: 10.7150/jca.20150

The phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in BANCR -silenced or -overexpressing NPA and TPC1 cells was detected by western blotting. The loss of BANCR induced the phosphorylation of ERK1/2 (C). However, there was no activation of upstream molecule including EGFR (A), MEK (C), MEK, STAT-1, and STAT-3 (E). BANCR overexpression had no effect on the phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in either cell line (B, D and F). GAPDH expression was used to normalize protein loading.
Figure Legend Snippet: The phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in BANCR -silenced or -overexpressing NPA and TPC1 cells was detected by western blotting. The loss of BANCR induced the phosphorylation of ERK1/2 (C). However, there was no activation of upstream molecule including EGFR (A), MEK (C), MEK, STAT-1, and STAT-3 (E). BANCR overexpression had no effect on the phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in either cell line (B, D and F). GAPDH expression was used to normalize protein loading.

Techniques Used: Western Blot, Activation Assay, Over Expression, Expressing

21) Product Images from "Triphala Suppresses Growth and Migration of Human Gastric Carcinoma Cells In Vitro and in a Zebrafish Xenograft Model"

Article Title: Triphala Suppresses Growth and Migration of Human Gastric Carcinoma Cells In Vitro and in a Zebrafish Xenograft Model

Journal: BioMed Research International

doi: 10.1155/2018/7046927

Analysis of the effect of Triphala on the EGFR signaling cascade. The Western blots compare lysates of untreated and Triphala-treated MGC803 cells probed with p-EGFR, p-ERK1/2, and p-AKT monoclonal antibodies as well as with an anti-GAPDH antibody as a control.
Figure Legend Snippet: Analysis of the effect of Triphala on the EGFR signaling cascade. The Western blots compare lysates of untreated and Triphala-treated MGC803 cells probed with p-EGFR, p-ERK1/2, and p-AKT monoclonal antibodies as well as with an anti-GAPDH antibody as a control.

Techniques Used: Western Blot

22) Product Images from "AXL confers intrinsic resistance to osimertinib and advances the emergence of tolerant cells"

Article Title: AXL confers intrinsic resistance to osimertinib and advances the emergence of tolerant cells

Journal: Nature Communications

doi: 10.1038/s41467-018-08074-0

Osimertinib activated AXL in EGFR -mutated NSCLC cells in vitro. a Human tyrosine kinase phosphorylation array analysis of PC-9 cells in the presence or absence of osimertinib (100 nmol/L) for 72 h. b PC-9 cells were treated with osimertinib (100 nmol/L), lysed, and the indicated proteins detected by western blotting. c Nonspecific siRNA control, specific siRNA for AXL , HER3 or MET introduced into the indicated cells. After 24 h, the cells were incubated with or without osimertinib (100 nmol/L) for 72 h and cell viability was determined using MTT assays. * P
Figure Legend Snippet: Osimertinib activated AXL in EGFR -mutated NSCLC cells in vitro. a Human tyrosine kinase phosphorylation array analysis of PC-9 cells in the presence or absence of osimertinib (100 nmol/L) for 72 h. b PC-9 cells were treated with osimertinib (100 nmol/L), lysed, and the indicated proteins detected by western blotting. c Nonspecific siRNA control, specific siRNA for AXL , HER3 or MET introduced into the indicated cells. After 24 h, the cells were incubated with or without osimertinib (100 nmol/L) for 72 h and cell viability was determined using MTT assays. * P

Techniques Used: In Vitro, Western Blot, Incubation, MTT Assay

AXL protein expression inversely correlated with susceptibility to EGFR-TKIs. a The EGFR -mutated NSCLC cell lines PC-9, PC-9GXR, PC-9KGR, H1650, H1975, HCC4011, HCC827, H3255, and HCC4006 were lysed and the indicated proteins were detected by western blotting. b The IC50 values for the EGFR-TKIs gefitinib and osimertinib in EGFR -mutated NSCLC cells. The IC50 values for both gefitinib and osimertinib were significantly higher in cells expressing high levels of AXL compared to cells expressing low levels of AXL. P values were calculated using the Mann Whitney U test. c Correlation between the cytoplasmic AXL protein expression levels determined immunohistochemically and the response to treatment with EGFR-TKIs in EGFR -mutated NSCLC specimens from 46 patients. d Correlation between the expression levels of the cytoplasmic AXL protein, determined immunohistochemically, and response to treatment with osimertinib in EGFR -mutated NSCLC specimens from 11 patients
Figure Legend Snippet: AXL protein expression inversely correlated with susceptibility to EGFR-TKIs. a The EGFR -mutated NSCLC cell lines PC-9, PC-9GXR, PC-9KGR, H1650, H1975, HCC4011, HCC827, H3255, and HCC4006 were lysed and the indicated proteins were detected by western blotting. b The IC50 values for the EGFR-TKIs gefitinib and osimertinib in EGFR -mutated NSCLC cells. The IC50 values for both gefitinib and osimertinib were significantly higher in cells expressing high levels of AXL compared to cells expressing low levels of AXL. P values were calculated using the Mann Whitney U test. c Correlation between the cytoplasmic AXL protein expression levels determined immunohistochemically and the response to treatment with EGFR-TKIs in EGFR -mutated NSCLC specimens from 46 patients. d Correlation between the expression levels of the cytoplasmic AXL protein, determined immunohistochemically, and response to treatment with osimertinib in EGFR -mutated NSCLC specimens from 11 patients

Techniques Used: Expressing, Western Blot, MANN-WHITNEY

AXL inhibitor sensitized AXL-high EGFR -mutant NSCLC cells to osimertinib. a PC-9, PC-9GXR, HCC4011, and HCC827 cells were incubated with osimertinib in the presence or absence of AXL inhibitor NPS1034 (1 μmol/L) for 72 h and the cell viability was determined using MTT assays. Data are representative of three independent experiments that produced similar results. b , c The indicated cells were incubated with osimertinib (100 nmol/L) with or without NPS1034 (1 μmol/L) for 1 h ( b ) and 72 h ( c ). The cells were lysed and the indicated proteins were detected by western blotting
Figure Legend Snippet: AXL inhibitor sensitized AXL-high EGFR -mutant NSCLC cells to osimertinib. a PC-9, PC-9GXR, HCC4011, and HCC827 cells were incubated with osimertinib in the presence or absence of AXL inhibitor NPS1034 (1 μmol/L) for 72 h and the cell viability was determined using MTT assays. Data are representative of three independent experiments that produced similar results. b , c The indicated cells were incubated with osimertinib (100 nmol/L) with or without NPS1034 (1 μmol/L) for 1 h ( b ) and 72 h ( c ). The cells were lysed and the indicated proteins were detected by western blotting

Techniques Used: Mutagenesis, Incubation, MTT Assay, Produced, Western Blot

AXL knockdown sensitized EGFR -mutated NSCLC cells to osimertinib in vitro. a Nonspecific siRNA control or AXL -specific siRNAs (#1 and #2) were introduced into PC-9, PC-9GXR, HCC4011, HCC827, and H3255 cells. After 24 h, the cells were incubated with osimertinib (100 nmol/L) or gefitinib (100 nmol/L) for 72 h and the cell growth was determined using MTT assays. The percentage of growth is shown relative to the growth of untreated control cells. b Quantitative determination of the inhibition of cell viability of high-AXL-expressing and low-AXL-expressing EGFR -mutant cells transfected with nonspecific siRNA control or AXL -specific siRNAs after treatment with osimertinib or gefitinib. Paired Student’s t tests were used for comparisons. c Nonspecific siRNA control or AXL -specific siRNAs were introduced into PC-9, PC-9GXR, and HCC4011 cells. After 24 h, the cells were incubated with osimertinib (100 nmol/L) for 1 h. The cells were lysed and the indicated proteins were detected by western blotting
Figure Legend Snippet: AXL knockdown sensitized EGFR -mutated NSCLC cells to osimertinib in vitro. a Nonspecific siRNA control or AXL -specific siRNAs (#1 and #2) were introduced into PC-9, PC-9GXR, HCC4011, HCC827, and H3255 cells. After 24 h, the cells were incubated with osimertinib (100 nmol/L) or gefitinib (100 nmol/L) for 72 h and the cell growth was determined using MTT assays. The percentage of growth is shown relative to the growth of untreated control cells. b Quantitative determination of the inhibition of cell viability of high-AXL-expressing and low-AXL-expressing EGFR -mutant cells transfected with nonspecific siRNA control or AXL -specific siRNAs after treatment with osimertinib or gefitinib. Paired Student’s t tests were used for comparisons. c Nonspecific siRNA control or AXL -specific siRNAs were introduced into PC-9, PC-9GXR, and HCC4011 cells. After 24 h, the cells were incubated with osimertinib (100 nmol/L) for 1 h. The cells were lysed and the indicated proteins were detected by western blotting

Techniques Used: In Vitro, Incubation, MTT Assay, Inhibition, Expressing, Mutagenesis, Transfection, Western Blot

23) Product Images from "HCRP-1 regulates EGFR–AKT–BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer"

Article Title: HCRP-1 regulates EGFR–AKT–BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1217-2

Hypothetic model of HCRP-1 depletion inhibits BIM expression via the EGFR–AKT signaling pathway
Figure Legend Snippet: Hypothetic model of HCRP-1 depletion inhibits BIM expression via the EGFR–AKT signaling pathway

Techniques Used: Expressing

Suppression of EGFR blocks the loss of HCRP-1 induced by the AKT signaling pathway, upregulates BIM expression, and suppresses anoikis resistance. a , b HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low-attachment surface. The whole-cell lysates were analyzed for the protein levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin by using western blot. c , d Cell anoikis was detected by flow cytometry. HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low- attachment surface. Cells that did not adhere were subjected to flow cytometry detection. e and f HCT116 and SW620 cells were co-transfected with HCRP-1 siRNA and/or EGFR siRNA. Levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin in cells that did not adhere were determined by Western blot. g , h Cell anoikis was detected by flow cytometry in colon cancer cells pretreated with HCRP-1 siRNA and/or EGFR siRNA. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P
Figure Legend Snippet: Suppression of EGFR blocks the loss of HCRP-1 induced by the AKT signaling pathway, upregulates BIM expression, and suppresses anoikis resistance. a , b HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low-attachment surface. The whole-cell lysates were analyzed for the protein levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin by using western blot. c , d Cell anoikis was detected by flow cytometry. HCT116 and SW620 cells were pretreated with HCRP-1 siRNA and the EGFR inhibitor, AG1478 (5 mM/L), and then suspended in six-well plates with low- attachment surface. Cells that did not adhere were subjected to flow cytometry detection. e and f HCT116 and SW620 cells were co-transfected with HCRP-1 siRNA and/or EGFR siRNA. Levels of p-EGFR, EGFR, p-AKT, AKT, BIM, and β-actin in cells that did not adhere were determined by Western blot. g , h Cell anoikis was detected by flow cytometry in colon cancer cells pretreated with HCRP-1 siRNA and/or EGFR siRNA. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P

Techniques Used: Expressing, Western Blot, Flow Cytometry, Cytometry, Transfection

Inhibition of HCRP-1 activates the EGFR/AKT signaling pathway and suppresses BIM protein expression. a , b HCT116 and SW620 cells were transfected with si-HCRP-1 or control siRNA, and then cells were harvested and submitted to Western blot detection for the protein expression of BIM, FoxO3a, EGFR, AKT, ERK, Mcl-1, Bcl-2, and β-actin. Phosphorylated forms of EGFR, AKT, FoxO3a, and ERK were also detected by western blot with the corresponding antibodies. c–f Cells were treatedwith CHX for 0, 0.5 , 1, and 2 h after transfection with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to western blot detection for the protein expression of EGFR. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P
Figure Legend Snippet: Inhibition of HCRP-1 activates the EGFR/AKT signaling pathway and suppresses BIM protein expression. a , b HCT116 and SW620 cells were transfected with si-HCRP-1 or control siRNA, and then cells were harvested and submitted to Western blot detection for the protein expression of BIM, FoxO3a, EGFR, AKT, ERK, Mcl-1, Bcl-2, and β-actin. Phosphorylated forms of EGFR, AKT, FoxO3a, and ERK were also detected by western blot with the corresponding antibodies. c–f Cells were treatedwith CHX for 0, 0.5 , 1, and 2 h after transfection with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to western blot detection for the protein expression of EGFR. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P

Techniques Used: Inhibition, Expressing, Transfection, Western Blot

24) Product Images from "EGFR Signaling Promotes TGFβ-Dependent Renal Fibrosis"

Article Title: EGFR Signaling Promotes TGFβ-Dependent Renal Fibrosis

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2011070645

Ang II–mediated ERK activation in AT1R/Cl4 cells is mediated by sustained EGFR activation. (A) ERK 1/2 phosphorylation increased in response to either Ang II or EGF, but only the Ang II–dependent activation was sustained. (B) Knockdown
Figure Legend Snippet: Ang II–mediated ERK activation in AT1R/Cl4 cells is mediated by sustained EGFR activation. (A) ERK 1/2 phosphorylation increased in response to either Ang II or EGF, but only the Ang II–dependent activation was sustained. (B) Knockdown

Techniques Used: Activation Assay

Ang II–mediated increases in ROS in AT1R/Cl4 cells activate Src kinase, which mediate EGFR/ERK activation and increase TGF β expression. (A) Ang II increased ROS production, measured by fluorescence intensity of 2′,7′-dichlorodihydrofluorescin,
Figure Legend Snippet: Ang II–mediated increases in ROS in AT1R/Cl4 cells activate Src kinase, which mediate EGFR/ERK activation and increase TGF β expression. (A) Ang II increased ROS production, measured by fluorescence intensity of 2′,7′-dichlorodihydrofluorescin,

Techniques Used: Activation Assay, Expressing, Fluorescence

Inhibition of EGFR attenuates Ang II–mediated TGF β expression in renal cortex. (A) Chronic administration of Ang II selectively increased expression of Y845 EGFR and phospho-ERK 1/2 , which were markedly attenuated in EGFR ptKO mice. Ang II–mediated
Figure Legend Snippet: Inhibition of EGFR attenuates Ang II–mediated TGF β expression in renal cortex. (A) Chronic administration of Ang II selectively increased expression of Y845 EGFR and phospho-ERK 1/2 , which were markedly attenuated in EGFR ptKO mice. Ang II–mediated

Techniques Used: Inhibition, Expressing, Mouse Assay

25) Product Images from "Inhibition of histone deacetylases sensitizes EGF receptor‐TK inhibitor‐resistant non‐small‐cell lung cancer cells to erlotinib in vitro and in vivo) Inhibition of histone deacetylases sensitizes EGF receptor‐TK inhibitor‐resistant non‐small‐cell lung cancer cells to erlotinib in vitro and in vivo"

Article Title: Inhibition of histone deacetylases sensitizes EGF receptor‐TK inhibitor‐resistant non‐small‐cell lung cancer cells to erlotinib in vitro and in vivo) Inhibition of histone deacetylases sensitizes EGF receptor‐TK inhibitor‐resistant non‐small‐cell lung cancer cells to erlotinib in vitro and in vivo

Journal: British Journal of Pharmacology

doi: 10.1111/bph.13961

YF454A plus erlotinib significantly suppresses the receptor tyrosine kinase signalling pathways in EGFR‐TKI‐resistant NSCLC cells. (A) Effects of either YF454A or erlotinib alone or in combination on the protein and mRNA expression of EGFR, Her2, AXL, c‐Met and IGF‐1R. PC9/ER cells were treated with YF454A (0.2 μM), erlotinib (5 μM) or YF454A/erlotinib for 24 h. Cell lysates and total mRNA were prepared, and Western blotting assays ( i ) and real‐time PCR analysis ( ii ) were further carried out. (B) The combination of YF454A and erlotinib time‐dependently suppressed protein expression ( i ) and mRNA expression ( ii ) of EGFR, HER2, AXL, MET and IGF‐1R. (C, D) YF454A/erlotinib down‐regulated the phosphorylation of Akt and ERK in PC9/ER cells (C) and in HCC827/ER cells (D). PC9/ER and HCC827/ER cells were treated with YF454A (0.2 μM) and erlotinib (5 μM) as a single agent alone or in combination for 24 h. The whole‐cell lysates were prepared and probed with specific Akt and ERK antibodies. Western blotting and real‐time PCR assays were performed three independent times, and representative images are shown.
Figure Legend Snippet: YF454A plus erlotinib significantly suppresses the receptor tyrosine kinase signalling pathways in EGFR‐TKI‐resistant NSCLC cells. (A) Effects of either YF454A or erlotinib alone or in combination on the protein and mRNA expression of EGFR, Her2, AXL, c‐Met and IGF‐1R. PC9/ER cells were treated with YF454A (0.2 μM), erlotinib (5 μM) or YF454A/erlotinib for 24 h. Cell lysates and total mRNA were prepared, and Western blotting assays ( i ) and real‐time PCR analysis ( ii ) were further carried out. (B) The combination of YF454A and erlotinib time‐dependently suppressed protein expression ( i ) and mRNA expression ( ii ) of EGFR, HER2, AXL, MET and IGF‐1R. (C, D) YF454A/erlotinib down‐regulated the phosphorylation of Akt and ERK in PC9/ER cells (C) and in HCC827/ER cells (D). PC9/ER and HCC827/ER cells were treated with YF454A (0.2 μM) and erlotinib (5 μM) as a single agent alone or in combination for 24 h. The whole‐cell lysates were prepared and probed with specific Akt and ERK antibodies. Western blotting and real‐time PCR assays were performed three independent times, and representative images are shown.

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Molecular characterization of EGFR‐TKI‐resistant NSCLC cells. (A) Cytotoxicity of erlotinib in PC9, PC9/ER, HCC827 and HCC827/ER cells was determined using the cell viability assays. Means from three independent experiments are shown with bars representing SEM ( n = 3 per group). (B) Characterization of phosphorylation status of tyrosine kinases including Her2, EGFR, Met, IGF1R, AXL and their downstream molecules Akt and ERK in PC9, PC9/ER, HCC827 and HCC827/ER cells respectively.
Figure Legend Snippet: Molecular characterization of EGFR‐TKI‐resistant NSCLC cells. (A) Cytotoxicity of erlotinib in PC9, PC9/ER, HCC827 and HCC827/ER cells was determined using the cell viability assays. Means from three independent experiments are shown with bars representing SEM ( n = 3 per group). (B) Characterization of phosphorylation status of tyrosine kinases including Her2, EGFR, Met, IGF1R, AXL and their downstream molecules Akt and ERK in PC9, PC9/ER, HCC827 and HCC827/ER cells respectively.

Techniques Used:

26) Product Images from "Hyperactivation of EGFR and downstream effector phospholipase D1 by oncogenic FAM83B"

Article Title: Hyperactivation of EGFR and downstream effector phospholipase D1 by oncogenic FAM83B

Journal: Oncogene

doi: 10.1038/onc.2013.293

Elevated FAM83B expression hyperactivates EGFR leading to increased PLD activity ( A and B ) 293T cells were transfected with expression constructs encoding EGFR and FLAG-FAM83B as indicated. Immunoprecipitation was performed using a FLAG, EGFR, or GFP (control) antibody, and precipitated proteins analyzed by Western analysis to determine the amount of EGFR bound to FAM83B. Discontiguous lanes are from the same membrane and the same exposure. ( C) ) Immunoprecipitation was performed by incubating lysates from MDA468 breast cancer cells with either 500 μL (1X) or 1000 μL (2X) of FAM83B hybridoma supernatant or IgG (control) antibody. Precipitated proteins were analyzed by Western analysis for EGFR and FAM83B. The lane labeled 83B control contains lysate from 293T cells transfected with a plasmid encoding FAM83B. (D) 293T cells were transfected with expression constructs encoding GFP (Vector), EGFR, FLAG-FAM83B, and FLAG-FAM83B-K230A as indicated. Immunoprecipitation was performed using a FLAG antibody, and precipitated proteins analyzed by Western analysis to determine the amount of EGFR bound to FAM83B. ( E ) HME1 cells expressing full-length (FL/WT) FAM83B or FAM83B with a site-directed mutation at K230A were assessed for AIG. ( F ) HME1 cells expressing GFP, FAM83B, or FAM83B-K230A were left untreated or stimulated with serum in the presence of 1-butanol-d10 to trap the PLD product as phosphatidylbutanol-d9 (PtdBuOH), which was detected by mass spectrometric analysis. Data is plotted as a ratio of PtdBuOH to phosphatidylmethanol (PtdMeOH), which was added as an internal standard ( G ) Western analysis of HME1 cells expressing GFP, FAM83B, or FAM83B-K230A for phospho-ERK1/2, ERK1/2, phospho-4E-BP1, phospho-S6K, and GAPDH.
Figure Legend Snippet: Elevated FAM83B expression hyperactivates EGFR leading to increased PLD activity ( A and B ) 293T cells were transfected with expression constructs encoding EGFR and FLAG-FAM83B as indicated. Immunoprecipitation was performed using a FLAG, EGFR, or GFP (control) antibody, and precipitated proteins analyzed by Western analysis to determine the amount of EGFR bound to FAM83B. Discontiguous lanes are from the same membrane and the same exposure. ( C) ) Immunoprecipitation was performed by incubating lysates from MDA468 breast cancer cells with either 500 μL (1X) or 1000 μL (2X) of FAM83B hybridoma supernatant or IgG (control) antibody. Precipitated proteins were analyzed by Western analysis for EGFR and FAM83B. The lane labeled 83B control contains lysate from 293T cells transfected with a plasmid encoding FAM83B. (D) 293T cells were transfected with expression constructs encoding GFP (Vector), EGFR, FLAG-FAM83B, and FLAG-FAM83B-K230A as indicated. Immunoprecipitation was performed using a FLAG antibody, and precipitated proteins analyzed by Western analysis to determine the amount of EGFR bound to FAM83B. ( E ) HME1 cells expressing full-length (FL/WT) FAM83B or FAM83B with a site-directed mutation at K230A were assessed for AIG. ( F ) HME1 cells expressing GFP, FAM83B, or FAM83B-K230A were left untreated or stimulated with serum in the presence of 1-butanol-d10 to trap the PLD product as phosphatidylbutanol-d9 (PtdBuOH), which was detected by mass spectrometric analysis. Data is plotted as a ratio of PtdBuOH to phosphatidylmethanol (PtdMeOH), which was added as an internal standard ( G ) Western analysis of HME1 cells expressing GFP, FAM83B, or FAM83B-K230A for phospho-ERK1/2, ERK1/2, phospho-4E-BP1, phospho-S6K, and GAPDH.

Techniques Used: Expressing, Activity Assay, Transfection, Construct, Immunoprecipitation, Western Blot, Labeling, Plasmid Preparation, Mutagenesis

Elevated PLD activity fails to recapitulate FAM83B phenotypes or rescue growth suppression following FAM83B ablation ( A ) HME1 cells expressing GFP or FAM83B were infected with retroviruses encoding cDNAs of GFP, PLD1, or PLD2 and AIG was assessed. ( B ) HME1 cells expressing RAS were infected with retroviruses encoding cDNAs of GFP, PLD1, or PLD2 . Subsequently, the resulting cell lines were infected with lentiviruses encoding shRNAs targeting GFP (G) or FAM83B (B) and cell growth was assessed 5 days later. ( C ) HME1 cells expressing GFP, PLD1, PLD2, or FAM83B were plated in the presence and absence of mammary epithelial growth supplement (MEGS) and cell number quantified 5 days later. ( D ) FAM83B confers decreased sensitivity to EGFR inhibition. HME1 cells expressing GFP, PLD1, PLD2, or FAM83B were treated with EGFR inhibitor AG1478 at 50, 100, and 200nM and cell number quantified 7 days later. ( E ) HME1 cells expressing GFP or FAM83B were grown as 3-dimensional (3D) cultures in lrBM in presence or absence of AG1478 (100 nM) for 10 days. Immunoblot analysis of phospho-Y1068-EGFR, total EGFR, phospho-ERK1/2, total ERK1/2, and GAPDH was performed. (F) Model of PLD involvement in FAM83B transformation and the pathways required.
Figure Legend Snippet: Elevated PLD activity fails to recapitulate FAM83B phenotypes or rescue growth suppression following FAM83B ablation ( A ) HME1 cells expressing GFP or FAM83B were infected with retroviruses encoding cDNAs of GFP, PLD1, or PLD2 and AIG was assessed. ( B ) HME1 cells expressing RAS were infected with retroviruses encoding cDNAs of GFP, PLD1, or PLD2 . Subsequently, the resulting cell lines were infected with lentiviruses encoding shRNAs targeting GFP (G) or FAM83B (B) and cell growth was assessed 5 days later. ( C ) HME1 cells expressing GFP, PLD1, PLD2, or FAM83B were plated in the presence and absence of mammary epithelial growth supplement (MEGS) and cell number quantified 5 days later. ( D ) FAM83B confers decreased sensitivity to EGFR inhibition. HME1 cells expressing GFP, PLD1, PLD2, or FAM83B were treated with EGFR inhibitor AG1478 at 50, 100, and 200nM and cell number quantified 7 days later. ( E ) HME1 cells expressing GFP or FAM83B were grown as 3-dimensional (3D) cultures in lrBM in presence or absence of AG1478 (100 nM) for 10 days. Immunoblot analysis of phospho-Y1068-EGFR, total EGFR, phospho-ERK1/2, total ERK1/2, and GAPDH was performed. (F) Model of PLD involvement in FAM83B transformation and the pathways required.

Techniques Used: Activity Assay, Expressing, Infection, Inhibition, Transformation Assay

27) Product Images from "Src inhibition blocks renal interstitial fibroblast activation and ameliorates renal fibrosis"

Article Title: Src inhibition blocks renal interstitial fibroblast activation and ameliorates renal fibrosis

Journal: Kidney international

doi: 10.1038/ki.2015.293

Src inhibition blocks EGFR phosphorylation in the kidney after UUO injury Lysates of kidney tissue collected at day 7 after sham and UUO injury with or without PP1 were subject to immunoblot analysis with specific antibodies against phospho-EGFR (Tyr1068), phospho-EGFR (Tyr845), total EGFR or α-Tubulin (a). Expression levels of indicated proteins were quantified by densitometry; phosphorylated EGFR Tyr1068 and Tyr845 were normalized with total EGFR and total EGFR was normalized with α-Tubulin (b–d). Data are represented as the mean ± SEM. Bars with different letters (a–b) are significantly different from one another ( P
Figure Legend Snippet: Src inhibition blocks EGFR phosphorylation in the kidney after UUO injury Lysates of kidney tissue collected at day 7 after sham and UUO injury with or without PP1 were subject to immunoblot analysis with specific antibodies against phospho-EGFR (Tyr1068), phospho-EGFR (Tyr845), total EGFR or α-Tubulin (a). Expression levels of indicated proteins were quantified by densitometry; phosphorylated EGFR Tyr1068 and Tyr845 were normalized with total EGFR and total EGFR was normalized with α-Tubulin (b–d). Data are represented as the mean ± SEM. Bars with different letters (a–b) are significantly different from one another ( P

Techniques Used: Inhibition, Expressing

28) Product Images from "CCR7 high expression leads to cetuximab resistance by cross-talking with EGFR pathway in PI3K/AKT signals in colorectal cancer"

Article Title: CCR7 high expression leads to cetuximab resistance by cross-talking with EGFR pathway in PI3K/AKT signals in colorectal cancer

Journal: American Journal of Cancer Research

doi:

Cetuximab insensitive patients expressed higher CCR7 in tumor tissue. A. Representative images of CCR7 expression of CRC tissues from patients received Cetuximab therapy, Case 1, case 2, case 3 were from PD (progressive disease) group, and case 4, case 5, case 6 was from PR (partial remission) group. (Magnification: × 400). B. 14 samples was analyzed in this part, compared to PR group, the CCR7 expression level was significantly higher in the PD group, data was showed as mean expression intensity ± SEM (* P = 0.014). C. Double immunofluorescent staining of CCR7 and EGFR in CRC tissues from PD group (Magnification: × 400).
Figure Legend Snippet: Cetuximab insensitive patients expressed higher CCR7 in tumor tissue. A. Representative images of CCR7 expression of CRC tissues from patients received Cetuximab therapy, Case 1, case 2, case 3 were from PD (progressive disease) group, and case 4, case 5, case 6 was from PR (partial remission) group. (Magnification: × 400). B. 14 samples was analyzed in this part, compared to PR group, the CCR7 expression level was significantly higher in the PD group, data was showed as mean expression intensity ± SEM (* P = 0.014). C. Double immunofluorescent staining of CCR7 and EGFR in CRC tissues from PD group (Magnification: × 400).

Techniques Used: Expressing, Staining

29) Product Images from "EGFR and myosin II inhibitors cooperate to suppress EGFR‐T790M‐mutant NSCLC cells), EGFR and myosin II inhibitors cooperate to suppress EGFR‐T790M‐mutant NSCLC cells"

Article Title: EGFR and myosin II inhibitors cooperate to suppress EGFR‐T790M‐mutant NSCLC cells), EGFR and myosin II inhibitors cooperate to suppress EGFR‐T790M‐mutant NSCLC cells

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2012.02.001

Blebbistatin functions to weaken EGFR‐MYH9 and EGFR‐β‐actin interactions. (A). The cell proliferation was determined by the MTS assay in H1975 cells treated with blebbistatin for 3 days. The proliferation of vehicle‐treated
Figure Legend Snippet: Blebbistatin functions to weaken EGFR‐MYH9 and EGFR‐β‐actin interactions. (A). The cell proliferation was determined by the MTS assay in H1975 cells treated with blebbistatin for 3 days. The proliferation of vehicle‐treated

Techniques Used: MTS Assay

The EGFR interactions with MYH9 and β‐actin occur in the nuclei of H1975 cells. (A) H322, H358, H1650, and H1975 cells were fractionated and the cytoplasmic and nuclear fractions were subjected to co‐IP using an anti‐EGFR
Figure Legend Snippet: The EGFR interactions with MYH9 and β‐actin occur in the nuclei of H1975 cells. (A) H322, H358, H1650, and H1975 cells were fractionated and the cytoplasmic and nuclear fractions were subjected to co‐IP using an anti‐EGFR

Techniques Used: Co-Immunoprecipitation Assay

MYH9 and β‐actin are identified as EGFR‐associated proteins. (A) Whole cell extracts from H322 and H1975 cells were subjected to co‐IP analysis using an anti‐EGFR antibody. The proteins were separated on SDS‐PAGE
Figure Legend Snippet: MYH9 and β‐actin are identified as EGFR‐associated proteins. (A) Whole cell extracts from H322 and H1975 cells were subjected to co‐IP analysis using an anti‐EGFR antibody. The proteins were separated on SDS‐PAGE

Techniques Used: Co-Immunoprecipitation Assay, SDS Page

30) Product Images from "EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells"

Article Title: EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2015.043

EP2 overexpression increases Src phosphorylation and activity. (A) EP2 overexpression increases Src phosphorylation. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-Src. Cell lysates were subjected to western blot analysis for p-Src (Tyr416). Membranes were stripped and reprobed for Src to confirm equal expression of Src. (B) EP2 overexpression increases endogenous Src activity. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-EGFR. Twenty-four hours after transfection, cells were incubated with or without PP2 for another 24 hours. Cell lysates were subjected to western blot analysis for p-EGFR (Tyr845). Membranes were stripped and reprobed for EGFR to confirm equal expression of EGFR. Additionally, cell lysates were subjected to western blot analysis for p-Src and total Src to examine the effect of PP2 on endogenous Src phosphorylation in HEK 293 cells.
Figure Legend Snippet: EP2 overexpression increases Src phosphorylation and activity. (A) EP2 overexpression increases Src phosphorylation. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-Src. Cell lysates were subjected to western blot analysis for p-Src (Tyr416). Membranes were stripped and reprobed for Src to confirm equal expression of Src. (B) EP2 overexpression increases endogenous Src activity. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-EGFR. Twenty-four hours after transfection, cells were incubated with or without PP2 for another 24 hours. Cell lysates were subjected to western blot analysis for p-EGFR (Tyr845). Membranes were stripped and reprobed for EGFR to confirm equal expression of EGFR. Additionally, cell lysates were subjected to western blot analysis for p-Src and total Src to examine the effect of PP2 on endogenous Src phosphorylation in HEK 293 cells.

Techniques Used: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Incubation

31) Product Images from "Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells"

Article Title: Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells

Journal: Protein & Cell

doi: 10.1007/s13238-016-0285-2

Inhibitory effects of MD-1 on the EGFR signaling pathway in HSC-T6 cells . (A) Immunostaining of EGFR-positive HSC-T6 cells showed MD-1 localization. Nuclei are stained with DAPI. Scale bar: 10 µm. (B) Similar to (A), but in EGFR-knockdown HSC-T6 cells. Scale bar: 10 µm. (C) Co-immunoprecipitation blots showed MD-1-Biotin was precipitated by EGFR in the MD-1-treated HSC-T6 cells, but not in the parental cells. (D) Representative Western blots to show the expression of EGFR, AKT, cyclin D1, and p-Smad. GAPDH was used as the loading control. Densitometry was performed to determine the relative expression levels normalized to GAPDH. * P
Figure Legend Snippet: Inhibitory effects of MD-1 on the EGFR signaling pathway in HSC-T6 cells . (A) Immunostaining of EGFR-positive HSC-T6 cells showed MD-1 localization. Nuclei are stained with DAPI. Scale bar: 10 µm. (B) Similar to (A), but in EGFR-knockdown HSC-T6 cells. Scale bar: 10 µm. (C) Co-immunoprecipitation blots showed MD-1-Biotin was precipitated by EGFR in the MD-1-treated HSC-T6 cells, but not in the parental cells. (D) Representative Western blots to show the expression of EGFR, AKT, cyclin D1, and p-Smad. GAPDH was used as the loading control. Densitometry was performed to determine the relative expression levels normalized to GAPDH. * P

Techniques Used: Immunostaining, Staining, Immunoprecipitation, Western Blot, Expressing

Inhibitory effects of MD-1 on DMN-induced hepatic fibrosis in rats . (A) The sectioned rat liver tissues were stained with Van Gieson staining reagent to show the proliferated collagen fibers (stained with red) segmented and surrounded pseudolobules in the positive control group, and MD-1 treatment significantly reduced the collagen fibers. Original magnification: 200×. (B) Top: Expression of p-EGFR, p-AKT, α-SMA, cyclin D1, and p-Smad in sectioned rat liver was examined by immunohistochemistry. The percentages of positive cells (stained with brown) were counted within five fields of view for each section using a 20× objective lens. Original magnification: 100×. Bottom: Statistics for p-EGFR, p-AKT, α-SMA, cyclin D1, and p-Smad. * P
Figure Legend Snippet: Inhibitory effects of MD-1 on DMN-induced hepatic fibrosis in rats . (A) The sectioned rat liver tissues were stained with Van Gieson staining reagent to show the proliferated collagen fibers (stained with red) segmented and surrounded pseudolobules in the positive control group, and MD-1 treatment significantly reduced the collagen fibers. Original magnification: 200×. (B) Top: Expression of p-EGFR, p-AKT, α-SMA, cyclin D1, and p-Smad in sectioned rat liver was examined by immunohistochemistry. The percentages of positive cells (stained with brown) were counted within five fields of view for each section using a 20× objective lens. Original magnification: 100×. Bottom: Statistics for p-EGFR, p-AKT, α-SMA, cyclin D1, and p-Smad. * P

Techniques Used: Staining, Positive Control, Expressing, Immunohistochemistry

32) Product Images from "Sortilin limits EGFR signaling by promoting its internalization in lung cancer"

Article Title: Sortilin limits EGFR signaling by promoting its internalization in lung cancer

Journal: Nature Communications

doi: 10.1038/s41467-017-01172-5

Sortilin expression decreased with the tumor aggressiveness. a Boxplot diagram represents the quantification (Hirsch index) of sortilin expression in human lung adenocarcinoma ( n = 78) and highlights the high expression of sortilin at low grade, as seen in representative images in b : grade I, well-differentiated; grade II, moderately differentiated; grade III, poorly differentiated. Magnification ×50, insets ×200. c Boxplot diagram of the percentage of Ki-67-positive nuclei, reflecting cancer cell proliferation, and tumor aggressiveness in the same patients shown in b with representative images d . Magnification ×50, insets ×200. e Kaplan−Meier curves of overall survival were constructed for the following groups of patients with high (black curve, n = 248) or low (orange curve, n = 425) SORT1 expression, using the online tool at kmplot.com. f In silico analysis of SORT1 expression in lung adenocarcinoma with high or low EGFR expression; data were obtained from the MSKCC cBioPortal for Cancer Genomics database or Genome Expression Omnibus. g Quantitative PCR analyses for SORT1 and EGFR expression in a cohort of patients ( n = 20) with high or low EGFR expression. h Kaplan−Meier curves showing the survival benefit provided by high- SORT1 expression (black curve, n = 32) relative to low SORT1 expression (orange curve, n = 30) on a subset patients with high- EGFR expression; data were obtained from the lung adenocarcinoma cohort in the MSKCC cBioPortal database ( n = 522). i In silico analysis for SORT1 expression in lung adenocarcinoma with or without EGFR TKI−sensitive mutations, using data from the MSKCC cBioPortal for Cancer Genomics database or Genome Expression Omnibus. For Kaplan–Meier curves, the log-rank (Mantel-cox) is used test to determine the statistical significance of association sortilin high or low expression and overall survival within lung adenocarcinoma patients, * P
Figure Legend Snippet: Sortilin expression decreased with the tumor aggressiveness. a Boxplot diagram represents the quantification (Hirsch index) of sortilin expression in human lung adenocarcinoma ( n = 78) and highlights the high expression of sortilin at low grade, as seen in representative images in b : grade I, well-differentiated; grade II, moderately differentiated; grade III, poorly differentiated. Magnification ×50, insets ×200. c Boxplot diagram of the percentage of Ki-67-positive nuclei, reflecting cancer cell proliferation, and tumor aggressiveness in the same patients shown in b with representative images d . Magnification ×50, insets ×200. e Kaplan−Meier curves of overall survival were constructed for the following groups of patients with high (black curve, n = 248) or low (orange curve, n = 425) SORT1 expression, using the online tool at kmplot.com. f In silico analysis of SORT1 expression in lung adenocarcinoma with high or low EGFR expression; data were obtained from the MSKCC cBioPortal for Cancer Genomics database or Genome Expression Omnibus. g Quantitative PCR analyses for SORT1 and EGFR expression in a cohort of patients ( n = 20) with high or low EGFR expression. h Kaplan−Meier curves showing the survival benefit provided by high- SORT1 expression (black curve, n = 32) relative to low SORT1 expression (orange curve, n = 30) on a subset patients with high- EGFR expression; data were obtained from the lung adenocarcinoma cohort in the MSKCC cBioPortal database ( n = 522). i In silico analysis for SORT1 expression in lung adenocarcinoma with or without EGFR TKI−sensitive mutations, using data from the MSKCC cBioPortal for Cancer Genomics database or Genome Expression Omnibus. For Kaplan–Meier curves, the log-rank (Mantel-cox) is used test to determine the statistical significance of association sortilin high or low expression and overall survival within lung adenocarcinoma patients, * P

Techniques Used: Expressing, Construct, In Silico, Real-time Polymerase Chain Reaction

Model of sortilin function in regulation of EGFR at the cell surface Representative scheme in which sortilin acts as a key regulator of EGFR retrograde transport. Sortilin is mainly localized at the trans-Golgi network (TGN), and the plasma membrane fraction of sortilin cycles continually between the cell surface and the TGN via the endosomes (1). Sortilin binds both unstimulated and stimulated EGFR to allow their internalization (2). Thus, EGFR undergoes intracellular trafficking and sortilin mediates its loading in intraluminal vesicles (3). This ultimately results either in EGFR release through exosomes (4) or receptor degradation (5), thus ensuring signal termination. Conversely, sortilin downregulation impairs EGFR internalization (6), and EGFR consequently retained at the cell surface. Moreover, in sortilin-downregulated cells, EGFR exists in a hyperphosphorylated state, and transduces a constitutive survival signal that promotes tumor growth
Figure Legend Snippet: Model of sortilin function in regulation of EGFR at the cell surface Representative scheme in which sortilin acts as a key regulator of EGFR retrograde transport. Sortilin is mainly localized at the trans-Golgi network (TGN), and the plasma membrane fraction of sortilin cycles continually between the cell surface and the TGN via the endosomes (1). Sortilin binds both unstimulated and stimulated EGFR to allow their internalization (2). Thus, EGFR undergoes intracellular trafficking and sortilin mediates its loading in intraluminal vesicles (3). This ultimately results either in EGFR release through exosomes (4) or receptor degradation (5), thus ensuring signal termination. Conversely, sortilin downregulation impairs EGFR internalization (6), and EGFR consequently retained at the cell surface. Moreover, in sortilin-downregulated cells, EGFR exists in a hyperphosphorylated state, and transduces a constitutive survival signal that promotes tumor growth

Techniques Used:

EGF promotes the EGFR—sortilin interaction. a A549 cells grown in complete cell culture media were stimulated or not with EGF (50 ng/mL) for 30 min. Immunoprecipitations (IP) were performed using anti-EGFR antibody, and the immunocomplexes were immunoblotted (IB) using anti-sortilin antibody (top). In parallel, immunoblots for P-EGFR, EGFR, sortilin, P-ERK, and ERK were performed on whole-cell lysates (WCL); the isotypic lane Immunoglobulin G (IgG) represents the IP control. b Proximity ligation assays (PLA) were performed on A549 cells, non-stimulated or stimulated with EGF (50 ng/mL) for 2, 5, 15, and 30 min. Red spots indicate sites of proximity ligation assay amplification, reflecting the EGFR—sortilin interaction (white arrows). Scale bar, 10 µm. c Quantification of PLA time course, in comparison with non-stimulated cells. d A549 cells were stimulated or not with EGF (50 ng/mL) for 30 min, and then co-immunolabeled for EGFR and markers of the early endosome (Rab5), the late endosome/lysosome (LAMP2) and the trans-Golgi network (TGN46). For sortilin labeling, A549 cells were transiently transfected with sortilin-GFP to adapt a set of functional antibodies, and then immunolabeled for the same markers. Scale bar, 5 µm. e Quantitative analysis of EGFR or sortilin-GFP co-localization with the aforementioned organelle-specific markers. f A549 cells were transiently transfected with sortilin-GFP, and then stimulated with EGF (50 ng/mL) for 30 min. Next, cells were fixed and co-immunolabeled for EGFR and Rab5. Scale bar, 10 µm. All values represent means ± SD, Student’s t -test * P
Figure Legend Snippet: EGF promotes the EGFR—sortilin interaction. a A549 cells grown in complete cell culture media were stimulated or not with EGF (50 ng/mL) for 30 min. Immunoprecipitations (IP) were performed using anti-EGFR antibody, and the immunocomplexes were immunoblotted (IB) using anti-sortilin antibody (top). In parallel, immunoblots for P-EGFR, EGFR, sortilin, P-ERK, and ERK were performed on whole-cell lysates (WCL); the isotypic lane Immunoglobulin G (IgG) represents the IP control. b Proximity ligation assays (PLA) were performed on A549 cells, non-stimulated or stimulated with EGF (50 ng/mL) for 2, 5, 15, and 30 min. Red spots indicate sites of proximity ligation assay amplification, reflecting the EGFR—sortilin interaction (white arrows). Scale bar, 10 µm. c Quantification of PLA time course, in comparison with non-stimulated cells. d A549 cells were stimulated or not with EGF (50 ng/mL) for 30 min, and then co-immunolabeled for EGFR and markers of the early endosome (Rab5), the late endosome/lysosome (LAMP2) and the trans-Golgi network (TGN46). For sortilin labeling, A549 cells were transiently transfected with sortilin-GFP to adapt a set of functional antibodies, and then immunolabeled for the same markers. Scale bar, 5 µm. e Quantitative analysis of EGFR or sortilin-GFP co-localization with the aforementioned organelle-specific markers. f A549 cells were transiently transfected with sortilin-GFP, and then stimulated with EGF (50 ng/mL) for 30 min. Next, cells were fixed and co-immunolabeled for EGFR and Rab5. Scale bar, 10 µm. All values represent means ± SD, Student’s t -test * P

Techniques Used: Cell Culture, Western Blot, Ligation, Proximity Ligation Assay, Amplification, Immunolabeling, Labeling, Transfection, Functional Assay

EGFR interacts with sortilin at the cell surface. a A549 cells were pretreated or not with the cell-permeable dynamin inhibitor Dynasore (40 µM) for 2 h, and then stimulated or not with EGF (50 ng/mL) for 15 min. Cells were immunolabeled for EGFR and the early endosome marker EEA1, and then analyzed by confocal microscopy. Scale bar, 10 µm. Inset 4–3: Bright-field image of A549 cells; white arrows show EGFR clusters at the cell surface. Scale bar, 10 µm. b A549 cells were pretreated or not with Dynasore (40 µM) for 2 h, and then stimulated or not with EGF (50 ng/mL) for 15 min. The cell lysates were analyzed by western blotting for P-EGFR and EGFR. c A549 cells were transfected with EEA1-GFP, pretreated with Dynasore (40 µM) for 2 h, then stimulated with EGF (50 ng/mL) for 30 min. Next, cells were co-immunolabeled for EGFR and sortilin. Scale bar, 10 µm. The co-localization profile is shown in d . e Proximity ligation assays (PLA) were performed on A549 cells under the same conditions described above in a . Scale bar, 10 µm. f PLA quantification in comparison with A549 non-stimulated cells. All values represent means ± SD, Student’s t -test *** P
Figure Legend Snippet: EGFR interacts with sortilin at the cell surface. a A549 cells were pretreated or not with the cell-permeable dynamin inhibitor Dynasore (40 µM) for 2 h, and then stimulated or not with EGF (50 ng/mL) for 15 min. Cells were immunolabeled for EGFR and the early endosome marker EEA1, and then analyzed by confocal microscopy. Scale bar, 10 µm. Inset 4–3: Bright-field image of A549 cells; white arrows show EGFR clusters at the cell surface. Scale bar, 10 µm. b A549 cells were pretreated or not with Dynasore (40 µM) for 2 h, and then stimulated or not with EGF (50 ng/mL) for 15 min. The cell lysates were analyzed by western blotting for P-EGFR and EGFR. c A549 cells were transfected with EEA1-GFP, pretreated with Dynasore (40 µM) for 2 h, then stimulated with EGF (50 ng/mL) for 30 min. Next, cells were co-immunolabeled for EGFR and sortilin. Scale bar, 10 µm. The co-localization profile is shown in d . e Proximity ligation assays (PLA) were performed on A549 cells under the same conditions described above in a . Scale bar, 10 µm. f PLA quantification in comparison with A549 non-stimulated cells. All values represent means ± SD, Student’s t -test *** P

Techniques Used: Immunolabeling, Marker, Confocal Microscopy, Western Blot, Transfection, Ligation, Proximity Ligation Assay

Loss of sortilin greatly perturbs EGFR internalization and dramatically promotes EGFR signaling. a A549 cells expressing shRNA targeting sortilin and control cells (pLKO) were stimulated with EGF (50 ng/mL) for 30 min, and then fixed and immunolabeled for EGFR. Immunofluorescence was analyzed by confocal microscopy. The ratio between whole-cell and intracytoplasmic EGFR intensities, reflecting EGFR membrane retention, is quantified in b . c Sortilin-depleted and control A549 cells (pLKO) were stimulated with EGF (50 ng/mL) over a 60 min time course. At each time point, cell surface EGFR was stained at 4 °C using Alexa Fluor 488 anti-EGFR, and then analyzed by flow cytometry. Curves represent mean fluorescence intensity in sortilin-depleted cells relative to the control. d Cell lysates from sortilin-depleted (sortilin shRNA) or control A549 cells (pLKO) were immunoblotted with the indicated antibodies. e A549 sortilin-depleted and control cells (pLKO) were stimulated with EGF (50 ng/mL) over a 60 min time course. Cell lysates were analyzed by western blotting for components of the canonical EGFR signaling pathway using the indicated antibodies. f Sortilin-depleted cells were transfected with control siRNA (Si co) or EGFR siRNA. Cell lysates were analyzed by western blotting with the indicated antibodies. g Representative histograms of cell proliferation, as determined by EdU incorporation. Sortilin-depleted cells transfected or not with EGFR siRNA and control A549 cells (pLKO) were stimulated with EGF (50 ng/mL) for 1 h, and then fixed and treated for EdU incorporation. Percentages of EdU-positive cells were calculated by flow-cytometric analysis. h Sortilin-depleted (sortilin shRNA) or control A549 cells (pLKO) were pretreated with bafilomycin A1 (BAFA1) or MG132 for 2 h, and then stimulated or not with EGF (50 ng/mL) for 30 min. Cell lysates were analyzed by western blotting for EGFR and sortilin protein expression. i Quantitative PCR analysis of EGFR expression in sortilin-depleted and control A549 cells (pLKO) with or without EGF stimulation (50 ng/mL for 30 min). Results are presented in terms of fold change after normalization against HPRT mRNA. j Cell lysates from H3255, H1650, and H1975 were analyzed for EGFR and sortilin protein expression. k H1975 cells were transfected or not with SORT1 overexpression vector, and cell lysates were then analyzed by western blotting for the indicated proteins. l Representative histograms of cell proliferation, as determined by EdU incorporation in sortilin-overexpressing and control H1975 cells. m Sortilin-overexpressing and control H1975 cells were treated with increasing doses of gefitinib for 24 h, and cell lysates were analyzed by western blotting for the indicated proteins. All values represent means ± SD, Student’s t -test *** P
Figure Legend Snippet: Loss of sortilin greatly perturbs EGFR internalization and dramatically promotes EGFR signaling. a A549 cells expressing shRNA targeting sortilin and control cells (pLKO) were stimulated with EGF (50 ng/mL) for 30 min, and then fixed and immunolabeled for EGFR. Immunofluorescence was analyzed by confocal microscopy. The ratio between whole-cell and intracytoplasmic EGFR intensities, reflecting EGFR membrane retention, is quantified in b . c Sortilin-depleted and control A549 cells (pLKO) were stimulated with EGF (50 ng/mL) over a 60 min time course. At each time point, cell surface EGFR was stained at 4 °C using Alexa Fluor 488 anti-EGFR, and then analyzed by flow cytometry. Curves represent mean fluorescence intensity in sortilin-depleted cells relative to the control. d Cell lysates from sortilin-depleted (sortilin shRNA) or control A549 cells (pLKO) were immunoblotted with the indicated antibodies. e A549 sortilin-depleted and control cells (pLKO) were stimulated with EGF (50 ng/mL) over a 60 min time course. Cell lysates were analyzed by western blotting for components of the canonical EGFR signaling pathway using the indicated antibodies. f Sortilin-depleted cells were transfected with control siRNA (Si co) or EGFR siRNA. Cell lysates were analyzed by western blotting with the indicated antibodies. g Representative histograms of cell proliferation, as determined by EdU incorporation. Sortilin-depleted cells transfected or not with EGFR siRNA and control A549 cells (pLKO) were stimulated with EGF (50 ng/mL) for 1 h, and then fixed and treated for EdU incorporation. Percentages of EdU-positive cells were calculated by flow-cytometric analysis. h Sortilin-depleted (sortilin shRNA) or control A549 cells (pLKO) were pretreated with bafilomycin A1 (BAFA1) or MG132 for 2 h, and then stimulated or not with EGF (50 ng/mL) for 30 min. Cell lysates were analyzed by western blotting for EGFR and sortilin protein expression. i Quantitative PCR analysis of EGFR expression in sortilin-depleted and control A549 cells (pLKO) with or without EGF stimulation (50 ng/mL for 30 min). Results are presented in terms of fold change after normalization against HPRT mRNA. j Cell lysates from H3255, H1650, and H1975 were analyzed for EGFR and sortilin protein expression. k H1975 cells were transfected or not with SORT1 overexpression vector, and cell lysates were then analyzed by western blotting for the indicated proteins. l Representative histograms of cell proliferation, as determined by EdU incorporation in sortilin-overexpressing and control H1975 cells. m Sortilin-overexpressing and control H1975 cells were treated with increasing doses of gefitinib for 24 h, and cell lysates were analyzed by western blotting for the indicated proteins. All values represent means ± SD, Student’s t -test *** P

Techniques Used: Expressing, shRNA, Immunolabeling, Immunofluorescence, Confocal Microscopy, Staining, Flow Cytometry, Cytometry, Fluorescence, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Over Expression, Plasmid Preparation

C-terminally truncated sortilin strongly interacts with EGFR at the cell surface independently of ligand stimulation. a HEK293 cells were transiently transfected with either full-length (FL) or C-terminally truncated (Δc) sortilin-GFP. Cells were fixed and immunolabeled for the trans-Golgi network marker TGN46, and then analyzed by confocal microscope. Scale bar, 10 µm. b Bars show the Mander’s coefficient, indicating that FL-sortilin-GFP co-localized with TGN46 to a greater degree than Δc-sortilin-GFP. c Bars show quantification of cell surface GFP intensity. d Strong interaction between EGFR and Δc-sortilin at the plasma membrane. HEK293 cells were transiently co-transfected with EGFR and FL- or Δc sortilin-GFP, and then fixed and immunolabeled for EGFR; immunofluorescence was analyzed by confocal microscopy. Scale bar, 10 µm. e HEK293 cells were transiently co-transfected with EGFR and FL or Δc sortilin-v5. Immunoprecipitations (IP) were performed using anti-v5 antibody, and immunocomplexes were analyzed by western blotting using anti-EGFR antibody. In parallel, immunoblots of EGFR and sortilin-v5 were performed on whole-cell lysate (WCL). f HEK293 cells were transiently co-transfected with EGFR-GFP and FL- or Δc sortilin. Next, cells were stimulated or not with EGF (50 ng/mL) for 30 min and immunoprecipitated using anti-GFP antibody. Immunocomplexes were analyzed by western blotting for sortilin. In parallel, immunoblots of P-EGFR, EGFR, P-ERK, and ERK were performed on WCL. g HEK293 cells were transiently co-transfected with ΔC sortilin and the myc-tagged extra- or intracellular domain (ECD and ICD, respectively). Next, cell lysates (CL) were immunoprecipitated (IP) with anti-sortilin and immunoblotted with anti-myc. h NSCLC cell lines harboring EGFR deletion (H1650) or point mutations (H3255, H1975) were stimulated or not with EGF (50 ng/mL) for 30 min. Cell lysates were immunoprecipitated (IP) using anti-sortilin, and immunocomplexes were analyzed by western blotting with anti-EGFR. i PLA quantification performed on NSCLC cell lines stimulated or not with EGF (50 ng/mL) for 30 min. j Proximity ligation assays (PLA) were performed on NSCLC cells under the same conditions as described in h . All values represent means ± SD, Student’s t -test *** P
Figure Legend Snippet: C-terminally truncated sortilin strongly interacts with EGFR at the cell surface independently of ligand stimulation. a HEK293 cells were transiently transfected with either full-length (FL) or C-terminally truncated (Δc) sortilin-GFP. Cells were fixed and immunolabeled for the trans-Golgi network marker TGN46, and then analyzed by confocal microscope. Scale bar, 10 µm. b Bars show the Mander’s coefficient, indicating that FL-sortilin-GFP co-localized with TGN46 to a greater degree than Δc-sortilin-GFP. c Bars show quantification of cell surface GFP intensity. d Strong interaction between EGFR and Δc-sortilin at the plasma membrane. HEK293 cells were transiently co-transfected with EGFR and FL- or Δc sortilin-GFP, and then fixed and immunolabeled for EGFR; immunofluorescence was analyzed by confocal microscopy. Scale bar, 10 µm. e HEK293 cells were transiently co-transfected with EGFR and FL or Δc sortilin-v5. Immunoprecipitations (IP) were performed using anti-v5 antibody, and immunocomplexes were analyzed by western blotting using anti-EGFR antibody. In parallel, immunoblots of EGFR and sortilin-v5 were performed on whole-cell lysate (WCL). f HEK293 cells were transiently co-transfected with EGFR-GFP and FL- or Δc sortilin. Next, cells were stimulated or not with EGF (50 ng/mL) for 30 min and immunoprecipitated using anti-GFP antibody. Immunocomplexes were analyzed by western blotting for sortilin. In parallel, immunoblots of P-EGFR, EGFR, P-ERK, and ERK were performed on WCL. g HEK293 cells were transiently co-transfected with ΔC sortilin and the myc-tagged extra- or intracellular domain (ECD and ICD, respectively). Next, cell lysates (CL) were immunoprecipitated (IP) with anti-sortilin and immunoblotted with anti-myc. h NSCLC cell lines harboring EGFR deletion (H1650) or point mutations (H3255, H1975) were stimulated or not with EGF (50 ng/mL) for 30 min. Cell lysates were immunoprecipitated (IP) using anti-sortilin, and immunocomplexes were analyzed by western blotting with anti-EGFR. i PLA quantification performed on NSCLC cell lines stimulated or not with EGF (50 ng/mL) for 30 min. j Proximity ligation assays (PLA) were performed on NSCLC cells under the same conditions as described in h . All values represent means ± SD, Student’s t -test *** P

Techniques Used: Transfection, Immunolabeling, Marker, Microscopy, Immunofluorescence, Confocal Microscopy, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Ligation

33) Product Images from "ANP promotes proliferation and inhibits apoptosis of ovarian granulosa cells by NPRA/PGRMC1/EGFR complex and improves ovary functions of PCOS rats"

Article Title: ANP promotes proliferation and inhibits apoptosis of ovarian granulosa cells by NPRA/PGRMC1/EGFR complex and improves ovary functions of PCOS rats

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.494

NPRA/PGRMC1/EGFR complex promoted the proliferation of ovarian granulosa cells through the MAPK/ERK signaling pathway. KGN cells were treated with RU486, or RU486 and ANP, respectively. ( a ) Immunoprecipitation (IP): anti-NPRA pulled down proteins. Immune blot (IB): levels of PGRMC1 and EGFR were detected by western blot. ( b ) IP: anti-PGRMC1 pulled down proteins. IB: levels of NPRA and EGFR were detected by western blot. ( c ) KGN cells were treated with RU486, RU486 and ANP, or the inhibitor of MAPK/ERK (PD58095). Phosphorylation of EGFR (p-EGFR) and ERK1/2 (p-ERK1/2) were detected by western blot. ( d ) Western blot analysis of PCNA expression in KGN cells. Statistical analysis was shown. Significance was indicated by * P
Figure Legend Snippet: NPRA/PGRMC1/EGFR complex promoted the proliferation of ovarian granulosa cells through the MAPK/ERK signaling pathway. KGN cells were treated with RU486, or RU486 and ANP, respectively. ( a ) Immunoprecipitation (IP): anti-NPRA pulled down proteins. Immune blot (IB): levels of PGRMC1 and EGFR were detected by western blot. ( b ) IP: anti-PGRMC1 pulled down proteins. IB: levels of NPRA and EGFR were detected by western blot. ( c ) KGN cells were treated with RU486, RU486 and ANP, or the inhibitor of MAPK/ERK (PD58095). Phosphorylation of EGFR (p-EGFR) and ERK1/2 (p-ERK1/2) were detected by western blot. ( d ) Western blot analysis of PCNA expression in KGN cells. Statistical analysis was shown. Significance was indicated by * P

Techniques Used: Aqueous Normal-phase Chromatography, Immunoprecipitation, Western Blot, Expressing

ANP activated AP1 and upregulated the expression of PCNA. KGN cells were treated with ANP, transfected with PGRMC1 siRNA, EGFR inhibitor (TKI), or ANP with PGRMC1 siRNA and TKI, respectively. ( a ) Western blot analysis of activation of AP1 (p-c-Fos, p-c-Jun). ( b ) Level of PCNA was detected by western blot. Significance was indicated by * P
Figure Legend Snippet: ANP activated AP1 and upregulated the expression of PCNA. KGN cells were treated with ANP, transfected with PGRMC1 siRNA, EGFR inhibitor (TKI), or ANP with PGRMC1 siRNA and TKI, respectively. ( a ) Western blot analysis of activation of AP1 (p-c-Fos, p-c-Jun). ( b ) Level of PCNA was detected by western blot. Significance was indicated by * P

Techniques Used: Aqueous Normal-phase Chromatography, Expressing, Transfection, Western Blot, Activation Assay

34) Product Images from "Autophagy processes are dependent on EGF receptor signaling"

Article Title: Autophagy processes are dependent on EGF receptor signaling

Journal: Oncotarget

doi: 10.18632/oncotarget.25708

Analysis of Beclin 1 protein interactions and Bcl2 and Bax protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.
Figure Legend Snippet: Analysis of Beclin 1 protein interactions and Bcl2 and Bax protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.

Techniques Used: Expressing, Western Blot

35) Product Images from "Exposure of Barrett’s and esophageal adenocarcinoma cells to Bile acids activates EGFR-STAT3 signaling axis via induction of APE1"

Article Title: Exposure of Barrett’s and esophageal adenocarcinoma cells to Bile acids activates EGFR-STAT3 signaling axis via induction of APE1

Journal: Oncogene

doi: 10.1038/s41388-018-0388-8

APE1 mediates bile salts-induced STAT3 activation via an EGFR-dependent mechanism Immunoblot analysis of CPB ( A ), FLO-1 ( B ) and OE33 ( C ) cells pretreated with EGFR inhibitor (Gefitinib, 25 μM) followed by exposure to acidic (pH4) bile salts (100 μM). The samples were analyzed for the indicated proteins, β-actin was used as an internal control. ( D ) Immunoblot analysis of OE33 cells with EGFR-knockdown via EGFR siRNA followed by treatment with acidic (pH4) bile salts (100 μM) for 20 minutes and allowed to recover in complete media. The samples were collected at 3 and 6h post recovery and analyzed for the indicated proteins, β-actin was used as an internal control. Immunoprecipitation (IP) of APE1 ( E ), EGFR ( F ) and STAT3 ( G ) in OE33 cells treated with acidic (pH4) bile salts (100 μM) and immunoblotted for the indicated proteins. Results shown are representative of at least three independent experiments.
Figure Legend Snippet: APE1 mediates bile salts-induced STAT3 activation via an EGFR-dependent mechanism Immunoblot analysis of CPB ( A ), FLO-1 ( B ) and OE33 ( C ) cells pretreated with EGFR inhibitor (Gefitinib, 25 μM) followed by exposure to acidic (pH4) bile salts (100 μM). The samples were analyzed for the indicated proteins, β-actin was used as an internal control. ( D ) Immunoblot analysis of OE33 cells with EGFR-knockdown via EGFR siRNA followed by treatment with acidic (pH4) bile salts (100 μM) for 20 minutes and allowed to recover in complete media. The samples were collected at 3 and 6h post recovery and analyzed for the indicated proteins, β-actin was used as an internal control. Immunoprecipitation (IP) of APE1 ( E ), EGFR ( F ) and STAT3 ( G ) in OE33 cells treated with acidic (pH4) bile salts (100 μM) and immunoblotted for the indicated proteins. Results shown are representative of at least three independent experiments.

Techniques Used: Activation Assay, Immunoprecipitation

APE1 co-localizes with EGFR and STAT3 (A and B) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3 with APE1. (C) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3. Protein interactions (red fluorescent signals) were revealed by PLA anti-rabbit plus probe and PLA anti-mouse minus probe in OE33 cells treated with acidic (pH4) bile salts (100 μM) as mentioned in the materials and methods. Nuclei were stained with DAPI (blue). Results shown are representative of at least three independent experiments.
Figure Legend Snippet: APE1 co-localizes with EGFR and STAT3 (A and B) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3 with APE1. (C) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3. Protein interactions (red fluorescent signals) were revealed by PLA anti-rabbit plus probe and PLA anti-mouse minus probe in OE33 cells treated with acidic (pH4) bile salts (100 μM) as mentioned in the materials and methods. Nuclei were stained with DAPI (blue). Results shown are representative of at least three independent experiments.

Techniques Used: In Situ, Proximity Ligation Assay, Staining

36) Product Images from "HCRP-1 regulates cell migration, invasion and angiogenesis via Src/ FAK signaling in human prostate cancer"

Article Title: HCRP-1 regulates cell migration, invasion and angiogenesis via Src/ FAK signaling in human prostate cancer

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.38112

Endogenous interaction of HCRP-1 and Src contributes to Src/FAK signaling pathway. A,B. Cells were treated CHX for 0, 0.5 h, 1h, 2h after transfected with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to Western blot detection for the protein expression of EGFR. C. Activities of EGFR, Src and FAK were determined by using Western blot analysis with antibodies specific for total and phosphorylated forms of EGFR, Src and FAK in silencing of HCRP-1 and control group for both PC3 and LnCap cell lines. D. Src were coprecipitated with HCRP-1 in whole-cell lysates of PC3 cells.
Figure Legend Snippet: Endogenous interaction of HCRP-1 and Src contributes to Src/FAK signaling pathway. A,B. Cells were treated CHX for 0, 0.5 h, 1h, 2h after transfected with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to Western blot detection for the protein expression of EGFR. C. Activities of EGFR, Src and FAK were determined by using Western blot analysis with antibodies specific for total and phosphorylated forms of EGFR, Src and FAK in silencing of HCRP-1 and control group for both PC3 and LnCap cell lines. D. Src were coprecipitated with HCRP-1 in whole-cell lysates of PC3 cells.

Techniques Used: Transfection, Western Blot, Expressing

37) Product Images from "Interaction of the EGF Receptor and the Hippo Pathway in the Diabetic Kidney"

Article Title: Interaction of the EGF Receptor and the Hippo Pathway in the Diabetic Kidney

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2015040415

Blocking EGFR expression or activation inhibited YAP protein expression and phosphorylation in cultured renal proximal tubule epithelial cells. (A) LLCPK-Cl4 cells were exposed to high glucose (Glu) for 24 hours after 3 days of control siRNA or EGFR-specific
Figure Legend Snippet: Blocking EGFR expression or activation inhibited YAP protein expression and phosphorylation in cultured renal proximal tubule epithelial cells. (A) LLCPK-Cl4 cells were exposed to high glucose (Glu) for 24 hours after 3 days of control siRNA or EGFR-specific

Techniques Used: Blocking Assay, Expressing, Activation Assay, Cell Culture

Upregulation of phospho-Akt, CTGF, and AREG expression in diabetic mice was blunted by administration of erlotinib or in EGFR ptKO mice. (A) Balb/c mice, 9–10 weeks old, were made diabetic with STZ and were given or not given erlotinib by gavage
Figure Legend Snippet: Upregulation of phospho-Akt, CTGF, and AREG expression in diabetic mice was blunted by administration of erlotinib or in EGFR ptKO mice. (A) Balb/c mice, 9–10 weeks old, were made diabetic with STZ and were given or not given erlotinib by gavage

Techniques Used: Expressing, Mouse Assay

EGFR activation–dependent increase of YAP protein expression and phosphorylation in nuclear and cytoplasm fractions in of renal epithelial cells in response to high glucose (Glu). (A) LLCPK-Cl4 cells were exposed to high glucose with or without
Figure Legend Snippet: EGFR activation–dependent increase of YAP protein expression and phosphorylation in nuclear and cytoplasm fractions in of renal epithelial cells in response to high glucose (Glu). (A) LLCPK-Cl4 cells were exposed to high glucose with or without

Techniques Used: Activation Assay, Expressing

EGFR dependence of increased YAP expression and phosphorylation in diabetic mouse proximal tubule epithelial cells. Wild-type balb/c mice 9–10 weeks old were subjected to five consecutive STZ injections followed by administration or no administration
Figure Legend Snippet: EGFR dependence of increased YAP expression and phosphorylation in diabetic mouse proximal tubule epithelial cells. Wild-type balb/c mice 9–10 weeks old were subjected to five consecutive STZ injections followed by administration or no administration

Techniques Used: Expressing, Mouse Assay

38) Product Images from "GRHL2 inhibits colorectal cancer progression and metastasis via oppressing epithelial-mesenchymal transition"

Article Title: GRHL2 inhibits colorectal cancer progression and metastasis via oppressing epithelial-mesenchymal transition

Journal: Cancer Biology & Therapy

doi: 10.1080/15384047.2019.1599664

WB analysis of EGFR/Ras/Raf/MAPK and AKT pathways in SW620 and HT29 cells.
Figure Legend Snippet: WB analysis of EGFR/Ras/Raf/MAPK and AKT pathways in SW620 and HT29 cells.

Techniques Used: Western Blot

39) Product Images from "Proline oxidase, a p53-induced gene, targets COX-2/PGE2 signaling to induce apoptosis and inhibit tumor growth in colorectal cancers"

Article Title: Proline oxidase, a p53-induced gene, targets COX-2/PGE2 signaling to induce apoptosis and inhibit tumor growth in colorectal cancers

Journal: Oncogene

doi: 10.1038/onc.2008.322

The involvement of epidermal growth factor receptor (EGFR) signaling. The DLD-1 Tet-Off proline oxidase (POX) cells were fed with medium without doxycycline (DOX) for 0, 1 and 3 days. The cells were harvested and cell lysates were prepared, western blotting was performed for phosphorylated-EGFR and EGFR ( a ). Cells were infected with Ad-manganese superoxide dismutase (MnSOD) or Ad-vector and 1 day later, DOX was removed from the medium for 3 days. Then the cells were harvested and cell lysates were prepared and western blotting for EGFR and its phosphorylated form were performed ( b ). ( c ) To determine the effect of EGF on POX-reduced cyclooxygenase-2 (COX-2) expression, DOX was removed from the medium for 3 days and the cells were treated with EGF at the concentrations of 10 and l00 ng/ml. Then the cells were harvested and cell lysates were prepared and western blotting for COX-2 was performed. The western blotting for actin was used as loading control.
Figure Legend Snippet: The involvement of epidermal growth factor receptor (EGFR) signaling. The DLD-1 Tet-Off proline oxidase (POX) cells were fed with medium without doxycycline (DOX) for 0, 1 and 3 days. The cells were harvested and cell lysates were prepared, western blotting was performed for phosphorylated-EGFR and EGFR ( a ). Cells were infected with Ad-manganese superoxide dismutase (MnSOD) or Ad-vector and 1 day later, DOX was removed from the medium for 3 days. Then the cells were harvested and cell lysates were prepared and western blotting for EGFR and its phosphorylated form were performed ( b ). ( c ) To determine the effect of EGF on POX-reduced cyclooxygenase-2 (COX-2) expression, DOX was removed from the medium for 3 days and the cells were treated with EGF at the concentrations of 10 and l00 ng/ml. Then the cells were harvested and cell lysates were prepared and western blotting for COX-2 was performed. The western blotting for actin was used as loading control.

Techniques Used: Western Blot, Infection, Plasmid Preparation, Expressing

40) Product Images from "Control of glioblastoma tumorigenesis by feed-forward cytokine signaling"

Article Title: Control of glioblastoma tumorigenesis by feed-forward cytokine signaling

Journal: Nature neuroscience

doi: 10.1038/nn.4295

The ligand OSM regulates the phosphorylation of EGFR and the pharmacological inhibitors of EGFRvIII phosphorylation impair EGFRvIII-OSMR interaction, ( a ) Immunohistochemical analyses of wild-type astrocytes, treated with 10 ng/ml of OSM, and assessed
Figure Legend Snippet: The ligand OSM regulates the phosphorylation of EGFR and the pharmacological inhibitors of EGFRvIII phosphorylation impair EGFRvIII-OSMR interaction, ( a ) Immunohistochemical analyses of wild-type astrocytes, treated with 10 ng/ml of OSM, and assessed

Techniques Used: Immunohistochemistry

Related Articles

Incubation:

Article Title: Manipulating the Lateral Diffusion of Surface-Anchored EGF Demonstrates that Receptor Clustering Modulates Phosphorylation Levels
Article Snippet: .. The anti-EGFR-pY-1068 primary rabbit IgG antibody (Cell Signaling Technologies, #3777S) was diluted at a 1:800 volume:volume ratio in 1% BSA (1X PBS) and added to each sample and incubated for 1h at RT. .. In experiments where the EGFR was also stained the anti-EGFR rat IgG primary antibody (Santa Cruz #71035) was incubated at the same dilution concurrently.

Article Title: Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment
Article Snippet: .. Next, IF buffer (0.2% (v/v) Triton X-100 + 0.1% (v/v) BSA (radioimmunoassay grade) + 0.05% Tween 20, 7.7 mM NaN3 in PBS) + 10% (v/v) goat serum was added into the channels and the devices were incubated at room temperature for 1.5 h. Later, primary antibodies, monoclonal Anti-α-Tubulin (1:500, T9026, Sigma-Aldrich), Ki-67 (1:100, (DSHB Hybridoma Product AFFN-KI67-3E6)), EGFR (1:1000, MA5-13319, Thermo Scientific), or pEGFR (1:100, 3777S, Cell Signaling Technology® ) were diluted at in IF buffer and devices were parafilmed and kept at 4 °C overnight. ..

other:

Article Title: EGF Receptor–Dependent YAP Activation Is Important for Renal Recovery from AKI
Article Snippet: Antibodies against EGFR (4060S), p-EGFR (3777S), YAP (14074S), TAZ (4883S), pan-TEAD (13295S), Laminin B2 (13823S), Cyclin D (2922S), p-Rb (9307S), Ki67 (9129S), phosphoinositide-dependent kinase 1 (PDK1; 3062S), p-Akt (4060S), Akt (2920S), and β -actin (4970S) were from Cell Signaling Technology (Beverly, MA).

RIA Assay:

Article Title: Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment
Article Snippet: .. Next, IF buffer (0.2% (v/v) Triton X-100 + 0.1% (v/v) BSA (radioimmunoassay grade) + 0.05% Tween 20, 7.7 mM NaN3 in PBS) + 10% (v/v) goat serum was added into the channels and the devices were incubated at room temperature for 1.5 h. Later, primary antibodies, monoclonal Anti-α-Tubulin (1:500, T9026, Sigma-Aldrich), Ki-67 (1:100, (DSHB Hybridoma Product AFFN-KI67-3E6)), EGFR (1:1000, MA5-13319, Thermo Scientific), or pEGFR (1:100, 3777S, Cell Signaling Technology® ) were diluted at in IF buffer and devices were parafilmed and kept at 4 °C overnight. ..

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    Cell Signaling Technology Inc p egfr
    Dephosphorylation of <t>EGFR</t> v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . <t>Akt</t> phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.
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    Dephosphorylation of EGFR v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . Akt phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.

    Journal: Cancer Medicine

    Article Title: Nimotuzumab enhances temozolomide‐induced growth suppression of glioma cells expressing mutant EGFR in vivo

    doi: 10.1002/cam4.614

    Figure Lengend Snippet: Dephosphorylation of EGFR v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . Akt phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.

    Article Snippet: Proteins on the PVDF membranes were probed with antibodies against EGFR (C13, BD Biosciences, San Jose, CA), P‐EGFR (Tyr1068), P‐EGFR (Tyr1173), Akt, P‐Akt (Thr308), P‐Akt (Ser473), P‐mammalian target of rapamycin (mTOR) (Ser2448), mTOR (Cell Signaling, Danvers, MA), O 6 ‐methylguanine‐DNA methyltransferase (MGMT) (MT3.1, Neo Markers, Fremont, CA), MSH6, MLH1, MSH2, PMS2 (BD Biosciences), detected by chemiluminescence, and quantified (ImageQuant LAS4010, GE Healthcare, Tokyo, Japan).

    Techniques: De-Phosphorylation Assay, In Vitro, Cell Culture, Western Blot, Expressing, Positive Control, Inhibition

    CX3CL1/CX3CR1 activates the Src/FAK pathway. (A) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was determined by western blotting. (B) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of EGFP, p-EGFR (Tyr992), PTK2B and p-PTK2B (Tyr402) was measured by western blotting. The expression of EGFR, p-EGFR (Tyr992), Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was measured by western blotting in CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (C) VCaP; (D) PC-3. PC-3 cells were pretreated with (E) bosutinib (0.5 nM for 3 h) or with (F) PF-562271 (0.2 nM for 0.5 h), following which CX3CL1 was added to cells (100 nM for 5, 15, 30, 45 or 60 min), and the expression of p-Src (Tyr416) and p-FAK (Tyr576/577) was examined. VCaP cells were pretreated with either afatinib (1 or 10 µ M for 4 h) or bosutinib (0.25 or 2 µ M for 1 h), following which CX3CL1 (100 nM) was added to cells (100 nM) for 30 min, and the expression of (G) Src, p-Src (Tyr416), (H) EGFR and p-EGFR (Tyr992) were detected by western blotting. (I and J) The expression of MMP-9 and ROCK1 was measured by western blotting CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (I) VCaP; (J) PC-3. Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; EGFR, epidermal growth factor receptor; p, phosphorylated; OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; PTK2B, protein-tyrosine kinase 2-β; CX3CR1, C-X3-C motif chemokine receptor 1; MMP-9, matrix metal-loproteinase-9; ROCK1, Rho-associated protein kinase 1.

    Journal: International Journal of Oncology

    Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

    doi: 10.3892/ijo.2018.4487

    Figure Lengend Snippet: CX3CL1/CX3CR1 activates the Src/FAK pathway. (A) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was determined by western blotting. (B) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of EGFP, p-EGFR (Tyr992), PTK2B and p-PTK2B (Tyr402) was measured by western blotting. The expression of EGFR, p-EGFR (Tyr992), Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was measured by western blotting in CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (C) VCaP; (D) PC-3. PC-3 cells were pretreated with (E) bosutinib (0.5 nM for 3 h) or with (F) PF-562271 (0.2 nM for 0.5 h), following which CX3CL1 was added to cells (100 nM for 5, 15, 30, 45 or 60 min), and the expression of p-Src (Tyr416) and p-FAK (Tyr576/577) was examined. VCaP cells were pretreated with either afatinib (1 or 10 µ M for 4 h) or bosutinib (0.25 or 2 µ M for 1 h), following which CX3CL1 (100 nM) was added to cells (100 nM) for 30 min, and the expression of (G) Src, p-Src (Tyr416), (H) EGFR and p-EGFR (Tyr992) were detected by western blotting. (I and J) The expression of MMP-9 and ROCK1 was measured by western blotting CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (I) VCaP; (J) PC-3. Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; EGFR, epidermal growth factor receptor; p, phosphorylated; OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; PTK2B, protein-tyrosine kinase 2-β; CX3CR1, C-X3-C motif chemokine receptor 1; MMP-9, matrix metal-loproteinase-9; ROCK1, Rho-associated protein kinase 1.

    Article Snippet: The membranes were incubated with specific antibodies, including CX3CL1 (1:1,000; cat. no. ab25088), CX3CR1 (1:1,000; cat. no. ab8021; Abcam), Rho-associated protein kinase 1 (ROCK1; 1:2,000; cat. no. ab45171), matrix metalloproteinase-9 (MMP-9; 1:1,000; cat. no. ab73734) (all from Abcam), FAK (1:1,000; cat. no. 3285), phosphorylated (p)-FAK (1:1,000; cat. no. 3281), Src (1:1,000; cat. no. 2108), p-Src (1:1,000; cat. no. 6943), EGFR (1:1,000; cat. no. 5616), p-EGFR (1:1,000; cat. no. 2235) [Cell Signaling Technology, Inc. (CST), Danvers, MA, USA] and GAPDH (1:5,000; cat. no. AF1186/AF0006; Beyotime Institute of Biotechnology) at 4°C overnight.

    Techniques: Expressing, Western Blot, Over Expression, Negative Control

    EGFR, Src and FAK inhibitors reverse cell migration induced by CX3CL1 in PC-3 cells. (A) Cell migration was measured by scratch wound assay. Representative images are presented (×200 magnification). (B) The results were summarized from three independent experiments. * P

    Journal: International Journal of Oncology

    Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

    doi: 10.3892/ijo.2018.4487

    Figure Lengend Snippet: EGFR, Src and FAK inhibitors reverse cell migration induced by CX3CL1 in PC-3 cells. (A) Cell migration was measured by scratch wound assay. Representative images are presented (×200 magnification). (B) The results were summarized from three independent experiments. * P

    Article Snippet: The membranes were incubated with specific antibodies, including CX3CL1 (1:1,000; cat. no. ab25088), CX3CR1 (1:1,000; cat. no. ab8021; Abcam), Rho-associated protein kinase 1 (ROCK1; 1:2,000; cat. no. ab45171), matrix metalloproteinase-9 (MMP-9; 1:1,000; cat. no. ab73734) (all from Abcam), FAK (1:1,000; cat. no. 3285), phosphorylated (p)-FAK (1:1,000; cat. no. 3281), Src (1:1,000; cat. no. 2108), p-Src (1:1,000; cat. no. 6943), EGFR (1:1,000; cat. no. 5616), p-EGFR (1:1,000; cat. no. 2235) [Cell Signaling Technology, Inc. (CST), Danvers, MA, USA] and GAPDH (1:5,000; cat. no. AF1186/AF0006; Beyotime Institute of Biotechnology) at 4°C overnight.

    Techniques: Migration, Scratch Wound Assay Assay

    Kinases associated with the CX3CL1/CX3CR1 axis. The association between kinases relevant to CX3CL1 in the Src/focal adhesion kinase signaling pathway were predicted using the Ingenuity Pathway Analysis database. Src, proto-oncogene tyrosine-protein kinase Src; PI3K, phosphatidylinositol 3-kinase; ITGB3, integrin β-3; EGFR, epidermal growth factor receptor; PTK2B, protein-tyrosine kinase 2-β.

    Journal: International Journal of Oncology

    Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

    doi: 10.3892/ijo.2018.4487

    Figure Lengend Snippet: Kinases associated with the CX3CL1/CX3CR1 axis. The association between kinases relevant to CX3CL1 in the Src/focal adhesion kinase signaling pathway were predicted using the Ingenuity Pathway Analysis database. Src, proto-oncogene tyrosine-protein kinase Src; PI3K, phosphatidylinositol 3-kinase; ITGB3, integrin β-3; EGFR, epidermal growth factor receptor; PTK2B, protein-tyrosine kinase 2-β.

    Article Snippet: The membranes were incubated with specific antibodies, including CX3CL1 (1:1,000; cat. no. ab25088), CX3CR1 (1:1,000; cat. no. ab8021; Abcam), Rho-associated protein kinase 1 (ROCK1; 1:2,000; cat. no. ab45171), matrix metalloproteinase-9 (MMP-9; 1:1,000; cat. no. ab73734) (all from Abcam), FAK (1:1,000; cat. no. 3285), phosphorylated (p)-FAK (1:1,000; cat. no. 3281), Src (1:1,000; cat. no. 2108), p-Src (1:1,000; cat. no. 6943), EGFR (1:1,000; cat. no. 5616), p-EGFR (1:1,000; cat. no. 2235) [Cell Signaling Technology, Inc. (CST), Danvers, MA, USA] and GAPDH (1:5,000; cat. no. AF1186/AF0006; Beyotime Institute of Biotechnology) at 4°C overnight.

    Techniques:

    Identification of EGFR phosphorylation sites involved in anti-CD9-induced apoptosis (20×). Twenty-four hrs of anti-CD9 antibody treatment was performed, cells were fixed and stained for activated EGFR Y845, 1068, 1086, 1148 and 1173 (green). Nuclei

    Journal:

    Article Title: Heparin Binding Epidermal Growth Factor-Like Growth Factor and PD169316 Prevent Apoptosis in Mouse Embryonic Stem Cells

    doi: 10.1093/jb/mvn153

    Figure Lengend Snippet: Identification of EGFR phosphorylation sites involved in anti-CD9-induced apoptosis (20×). Twenty-four hrs of anti-CD9 antibody treatment was performed, cells were fixed and stained for activated EGFR Y845, 1068, 1086, 1148 and 1173 (green). Nuclei

    Article Snippet: The antibodies against ERK 1/2, phosphorylated ERK 1/2, SAPK/JNK, phosphorylated SAPK/JNK, p38 MAPK, phosphorylated p38 MAPK, SRC, phosphorylated SRC, PLCγ, c-CBL, GRB, cleaved caspase-3, phosphorylated EGFR (Tyr 845, 1068 and 1173) and PY-100 were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Staining

    Phosphorylation of EGFR is suppressed by HB-EGF and p38 MAPK inhibitor (20×). After 24 h of anti-CD9 antibody treatment and fixation cells were stained for activated EGFR 1148 and 1173 (green). Nuclei are labelled with DAPI. Anti-CD9 antibody

    Journal:

    Article Title: Heparin Binding Epidermal Growth Factor-Like Growth Factor and PD169316 Prevent Apoptosis in Mouse Embryonic Stem Cells

    doi: 10.1093/jb/mvn153

    Figure Lengend Snippet: Phosphorylation of EGFR is suppressed by HB-EGF and p38 MAPK inhibitor (20×). After 24 h of anti-CD9 antibody treatment and fixation cells were stained for activated EGFR 1148 and 1173 (green). Nuclei are labelled with DAPI. Anti-CD9 antibody

    Article Snippet: The antibodies against ERK 1/2, phosphorylated ERK 1/2, SAPK/JNK, phosphorylated SAPK/JNK, p38 MAPK, phosphorylated p38 MAPK, SRC, phosphorylated SRC, PLCγ, c-CBL, GRB, cleaved caspase-3, phosphorylated EGFR (Tyr 845, 1068 and 1173) and PY-100 were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Staining

    CAY increased cAMP/PKA and Src/EGFR activation in papillomas and surrounding skin. In (B–E), skin or two to three papillomas from three individual mice were combined (six to nine papillomas total) for analysis, and each experiment was repeated.

    Journal:

    Article Title: The prostaglandin receptor EP2 activates multiple signaling pathways and ?-arrestin1 complex formation during mouse skin papilloma development

    doi: 10.1093/carcin/bgp168

    Figure Lengend Snippet: CAY increased cAMP/PKA and Src/EGFR activation in papillomas and surrounding skin. In (B–E), skin or two to three papillomas from three individual mice were combined (six to nine papillomas total) for analysis, and each experiment was repeated.

    Article Snippet: Antibodies for AKT, p-AKT (Ser473), ERK1/2, p-ERK1/2 (Thr202/Tyr204), Src and p-Src (Tyr416), EGFR and p-EGFR (Tyr845) and p-CREB (Ser133) were obtained from Cell Signaling Technology (Danvers, MA), and the H-Ras antibody was purchased from BD Biosciences (San Jose, CA).

    Techniques: Activation Assay, Mouse Assay

    Comparison of TPA-induced PKA and EGFR signaling in surrounding skin and papillomas and the effects of their inhibition on papilloma formation. ( A ) Differences in p-CREB (Ser133), EGFR, p-EGFR (Tyr845), H-Ras, p-Src (Tyr416), p-AKT (Ser473) and p-ERK1/2

    Journal:

    Article Title: The prostaglandin receptor EP2 activates multiple signaling pathways and ?-arrestin1 complex formation during mouse skin papilloma development

    doi: 10.1093/carcin/bgp168

    Figure Lengend Snippet: Comparison of TPA-induced PKA and EGFR signaling in surrounding skin and papillomas and the effects of their inhibition on papilloma formation. ( A ) Differences in p-CREB (Ser133), EGFR, p-EGFR (Tyr845), H-Ras, p-Src (Tyr416), p-AKT (Ser473) and p-ERK1/2

    Article Snippet: Antibodies for AKT, p-AKT (Ser473), ERK1/2, p-ERK1/2 (Thr202/Tyr204), Src and p-Src (Tyr416), EGFR and p-EGFR (Tyr845) and p-CREB (Ser133) were obtained from Cell Signaling Technology (Danvers, MA), and the H-Ras antibody was purchased from BD Biosciences (San Jose, CA).

    Techniques: Inhibition