Journal: Cell Death & Disease

Article Title: cPLA2 α mediates TGF- β -induced epithelial–mesenchymal transition in breast cancer through PI3k/Akt signaling

doi: 10.1038/cddis.2017.152

Figure Lengend Snippet: cPLA2 α were overexpressed in breast cancer tissues and invasive breast cancer cell lines. ( a ) A – Immunohistochemistry of cPLA2 α in mammary gland flocculus hyperplasia tissues; B – immunohistochemistry of cPLA2 α in breast cancer tissues with different histological grades; c – immunohistochemistry of cPLA2 α in primary breast cancer tissues and distant metastasis tumor tissues. ( b ) Kaplan–Meier overall survival analysis of 126 patients with high or low cPLA2 α expression. ( c ) Kaplan–Meier disease-free survival analysis of 126 patients with high or low cPLA2 α expression. ( d ) Western blot of cPLA2 α and p-cPLA2 α in four pairs of fresh-frozen tissues containing immunohistochemical cancerous tissues and non-cancerous tissues and one pair of primary cancerous tissues and metastasis tissues from breast cancer patient (C: cancer; N: non-cancer; P: primary; M: metastases). Subtypes of different breast cancer patients: Patient 1 (HER2-enriched), Patient 2 (triple negative), Patient 3 (triple negative), Patient 4 (luminal A) and Patient 5 (luminal A). ( e ) The protein level of cPLA2 α and p-cPLA2 α in different breast cancer cell lines and normal mammary epithelial cell line MCF-10A. Subtypes of different breast cancer cell lines: MCF-7 (luminal A); T47D (luminal A); Bcap-37 (luminal A); SK-BR-3 (HER2-enriched); MDA-MB-231 (triple negative); ZR-75-30 (triple negative)). Scale bar, 1.0 mm

Article Snippet: Antibodies against the following proteins were used: E-cadherin (1:1000) from BD Biosciences (San Jose, CA, USA); GAPDH (1:1000), N-cadherin (1:500), ZEB1 (1:100), Ovol2 (1:200) and Slug (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); vimentin (1:8000) from Epitomics (Burlingame, CA, USA); α -SMA (0.2 mg/ml) and Twist (1:1000) from Abcam (Hong Kong, China); Ovol1 (1:200) from Proteintech group (Chicago, IL, USA); cPLA2 α (1:1000) from GeneTex (Irvine, TX, USA); Phospho-cPLA2 α (1:1000), phospho-Akt (Ser473) (1:1000), phospho-FAK (1:1000), phospho-GSK-3 β (1:1000) Akt (Ser473)(1:1000), FAK (1:1000) and GSK-3 β (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibody from Santa Cruz Biotechnology (1:4000).

Techniques: Immunohistochemistry, Expressing, Western Blot, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: cPLA2 α mediates TGF- β -induced epithelial–mesenchymal transition in breast cancer through PI3k/Akt signaling

doi: 10.1038/cddis.2017.152

Figure Lengend Snippet: Clinicopathological characteristics of patients with different cPLA2 α expression levels undergoing surgery for breast cancer

Article Snippet: Antibodies against the following proteins were used: E-cadherin (1:1000) from BD Biosciences (San Jose, CA, USA); GAPDH (1:1000), N-cadherin (1:500), ZEB1 (1:100), Ovol2 (1:200) and Slug (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); vimentin (1:8000) from Epitomics (Burlingame, CA, USA); α -SMA (0.2 mg/ml) and Twist (1:1000) from Abcam (Hong Kong, China); Ovol1 (1:200) from Proteintech group (Chicago, IL, USA); cPLA2 α (1:1000) from GeneTex (Irvine, TX, USA); Phospho-cPLA2 α (1:1000), phospho-Akt (Ser473) (1:1000), phospho-FAK (1:1000), phospho-GSK-3 β (1:1000) Akt (Ser473)(1:1000), FAK (1:1000) and GSK-3 β (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibody from Santa Cruz Biotechnology (1:4000).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: cPLA2 α mediates TGF- β -induced epithelial–mesenchymal transition in breast cancer through PI3k/Akt signaling

doi: 10.1038/cddis.2017.152

Figure Lengend Snippet: cPLA2 α overexpression enhanced breast cancer cells migration and invasion in vitro . ( a ) Assessment of the transfected efficiency of cPLA2 α protein expression after retroviral infection in T47D cells. ( b ) Scratch assay comparing the migration of SCR/T47D (left) and overcPLA2 α /T47D (right) cells. ( c ) Comparison of chemotaxis potential of SCR/T47D (left) and overcPLA2 α /T47D (right) cells under the application of high concentration of FBS (20%) with constant stimulation for 6 h. ( d ) Comparison of the invasion potential of SCR/T47D (left) and overcPLA2 α /T47D (right) cells after incubation with EGF (10 ng/ml) at 37 °C in 5% CO 2 for 36 h by counting the number of the cells that invaded through Matrigel-coated transwell inserts. ** P <0.01, *** P <0.001. Scale bar, 1.0 mm. All experiments were repeated at least three times

Article Snippet: Antibodies against the following proteins were used: E-cadherin (1:1000) from BD Biosciences (San Jose, CA, USA); GAPDH (1:1000), N-cadherin (1:500), ZEB1 (1:100), Ovol2 (1:200) and Slug (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); vimentin (1:8000) from Epitomics (Burlingame, CA, USA); α -SMA (0.2 mg/ml) and Twist (1:1000) from Abcam (Hong Kong, China); Ovol1 (1:200) from Proteintech group (Chicago, IL, USA); cPLA2 α (1:1000) from GeneTex (Irvine, TX, USA); Phospho-cPLA2 α (1:1000), phospho-Akt (Ser473) (1:1000), phospho-FAK (1:1000), phospho-GSK-3 β (1:1000) Akt (Ser473)(1:1000), FAK (1:1000) and GSK-3 β (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibody from Santa Cruz Biotechnology (1:4000).

Techniques: Over Expression, Migration, In Vitro, Transfection, Expressing, Retroviral, Infection, Wound Healing Assay, Comparison, Chemotaxis Assay, Concentration Assay, Incubation

Journal: Cell Death & Disease

Article Title: cPLA2 α mediates TGF- β -induced epithelial–mesenchymal transition in breast cancer through PI3k/Akt signaling

doi: 10.1038/cddis.2017.152

Figure Lengend Snippet: cPLA2 α regulated MDA-MB-231 cell migration and invasion capacities in vitro . ( a ) Assessment of the transfected efficiency of cPLA2 α protein expression after retroviral infection in MDA-MB-231 cells. ( b ) Repression specificity of cPLA2 α mRNA expression in MDA-MB-231 cells by qRT-PCR. ( c ) Scratch assay comparing the migration of SCR/MDA-MB-231, sicPLA2 α /MDA-MB-231 and overcPLA2 α /MDA-MB-231 cells. ( d ) Comparison of chemotaxis potential of SCR/MDA-MB-231, sicPLA2 α MDA-MB-231 and overcPLA2 α /MDA-MB-231 cells under the application of high concentration of FBS (20%) with constant stimulation for 4 h. ( e ) Comparison of the invasion potential of SCR/MDA-MB-231, sicPLA2 α /MDA-MB-231 and overcPLA2 α /MDA-MB-231 cells after incubation with EGF (10 ng/ml) at 37 °C in 5% CO 2 for 24 h by counting the number of the cells that invaded through Matrigel-coated transwell inserts. ( f ) Comparison of the proliferation of SCR/MDA-MB-231, sicPLA2 α /MDA-MB-231 and overcPLA2 α /MDA-MB-231 cells by Cell Counting Kit-8 (CCK8) assay. ** P <0.01 and *** P <0.001. Scale bar, 1.0 mm. All experiments were repeated at least three times

Article Snippet: Antibodies against the following proteins were used: E-cadherin (1:1000) from BD Biosciences (San Jose, CA, USA); GAPDH (1:1000), N-cadherin (1:500), ZEB1 (1:100), Ovol2 (1:200) and Slug (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); vimentin (1:8000) from Epitomics (Burlingame, CA, USA); α -SMA (0.2 mg/ml) and Twist (1:1000) from Abcam (Hong Kong, China); Ovol1 (1:200) from Proteintech group (Chicago, IL, USA); cPLA2 α (1:1000) from GeneTex (Irvine, TX, USA); Phospho-cPLA2 α (1:1000), phospho-Akt (Ser473) (1:1000), phospho-FAK (1:1000), phospho-GSK-3 β (1:1000) Akt (Ser473)(1:1000), FAK (1:1000) and GSK-3 β (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibody from Santa Cruz Biotechnology (1:4000).

Techniques: Migration, In Vitro, Transfection, Expressing, Retroviral, Infection, Quantitative RT-PCR, Wound Healing Assay, Comparison, Chemotaxis Assay, Concentration Assay, Incubation, Cell Counting, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: cPLA2 α mediates TGF- β -induced epithelial–mesenchymal transition in breast cancer through PI3k/Akt signaling

doi: 10.1038/cddis.2017.152

Figure Lengend Snippet: The depression of cPLA2 α inhibited TGF- β -induced EMT and promoted MDA-MB-231 cells occurring MET. ( a ) Morphologic change of the MDA-MB-231 cells when cPLA2 α was endogenously knocked down. ( b ) Western blot of the expression of EMT-associated markers (epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and α -SMA) in MDA-MB-231 cells when cPLA2 α was knocked down or overexpressed, respectively. ( c ) Immunofluorescence staining for EMT-associated markers (epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin) in SCR/MDA-MB-231, sicPLA2 α /MDA-MB-231 and ovecPLA2 α /MDA-MB-231 cells, respectively. Scale bar, 20 μ m. All experiments were repeated at least three times. ( d ) Compared EMT-associated transcription factors (Slug, Twist and ZEB1) and MET-associated transcription factors (OVOL1 and OVOL2) expression between siSCR/MDA-MB-231 and sicPLA2 α /MDA-MB-231 cells (left); overSCR/MDA-MB-231 and overcPLA2 α /MDA-MB-231 cells (right) by western blot assay, respectively. Scale bar, 20 μ m

Article Snippet: Antibodies against the following proteins were used: E-cadherin (1:1000) from BD Biosciences (San Jose, CA, USA); GAPDH (1:1000), N-cadherin (1:500), ZEB1 (1:100), Ovol2 (1:200) and Slug (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); vimentin (1:8000) from Epitomics (Burlingame, CA, USA); α -SMA (0.2 mg/ml) and Twist (1:1000) from Abcam (Hong Kong, China); Ovol1 (1:200) from Proteintech group (Chicago, IL, USA); cPLA2 α (1:1000) from GeneTex (Irvine, TX, USA); Phospho-cPLA2 α (1:1000), phospho-Akt (Ser473) (1:1000), phospho-FAK (1:1000), phospho-GSK-3 β (1:1000) Akt (Ser473)(1:1000), FAK (1:1000) and GSK-3 β (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibody from Santa Cruz Biotechnology (1:4000).

Techniques: Western Blot, Expressing, Marker, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: cPLA2 α mediates TGF- β -induced epithelial–mesenchymal transition in breast cancer through PI3k/Akt signaling

doi: 10.1038/cddis.2017.152

Figure Lengend Snippet: Activation of cPLA2 α mediated TGF- β -induced EMT through the PI3K/Akt/GSK-3 β pathway. Western blot of phosphorylated cPLA2 α , FAK, Akt, GSK-3 β and total cPLA2 α , FAK, Akt, GSK-3 β in ( a ) siSCR/MDA-MB-231 and sicPLA2 α /MDA-MB-231 cells when cultured without or with TGF- β (5 ng/ml, 72 h); ( b ) overSCR/MDA-MB-231 and overcPLA2 α /MDA-MB-231 cells when cultured without or with TGF- β (5 ng/ml, 72 h); western blot of phosphorylated cPLA2 α , FAK, Akt, GSK-3 β ; total cPLA2 α , FAK, Akt, GSK-3 β and EMT-associated markers and transcription factors in ( c ) overcPLA2 α /MDA-MB-231 cells when cultured without or with TGF- β (5 ng/ml, 72 h) and LY294002; ( d ) overcPLA2 α /T47D cells when cultured without or with TGF- β (5 ng ng/ml, 72 h) and LY294002. All experiments were repeated at least three times

Article Snippet: Antibodies against the following proteins were used: E-cadherin (1:1000) from BD Biosciences (San Jose, CA, USA); GAPDH (1:1000), N-cadherin (1:500), ZEB1 (1:100), Ovol2 (1:200) and Slug (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); vimentin (1:8000) from Epitomics (Burlingame, CA, USA); α -SMA (0.2 mg/ml) and Twist (1:1000) from Abcam (Hong Kong, China); Ovol1 (1:200) from Proteintech group (Chicago, IL, USA); cPLA2 α (1:1000) from GeneTex (Irvine, TX, USA); Phospho-cPLA2 α (1:1000), phospho-Akt (Ser473) (1:1000), phospho-FAK (1:1000), phospho-GSK-3 β (1:1000) Akt (Ser473)(1:1000), FAK (1:1000) and GSK-3 β (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibody from Santa Cruz Biotechnology (1:4000).

Techniques: Activation Assay, Western Blot, Cell Culture

Journal: Cell Death & Disease

Article Title: cPLA2 α mediates TGF- β -induced epithelial–mesenchymal transition in breast cancer through PI3k/Akt signaling

doi: 10.1038/cddis.2017.152

Figure Lengend Snippet: Knockdown of cPLA2 α in MDA-MB-231 cells suppressed tumorigenesis and inhibited metastasis in vivo in lung cancer cells. Tumors from the female nude mice were orthotopically transplanted with the same number of MDA-MB-231 and sicPLA2 α /MDA-MB-231 cells (5 × 10 6 /mouse) into the fat pad ( n =10/group), and after 5 weeks, all the mice were killed and then were evaluated for ( a ) the primary tumors' volume, weight and growth rates for two groups and ( b ) the number of lung metastasis for two groups. ( c ) Western blot of the expression of EMT-associated markers (epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and α -SMA) in primary tumors for two groups. ( d ) Western blot of EMT-associated markers expression (epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and α -SMA) in metastases for two groups. ( e ) Immunohistochemistry of EMT-associated markers expression for two groups. ( f ) Immunofluorescence of the expression of EMT-associated markers for two groups. Data are represented as the mean±S.D., n =10. ** P <0.01 and *** P <0.001. Scale bar, 1.0 mm

Article Snippet: Antibodies against the following proteins were used: E-cadherin (1:1000) from BD Biosciences (San Jose, CA, USA); GAPDH (1:1000), N-cadherin (1:500), ZEB1 (1:100), Ovol2 (1:200) and Slug (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); vimentin (1:8000) from Epitomics (Burlingame, CA, USA); α -SMA (0.2 mg/ml) and Twist (1:1000) from Abcam (Hong Kong, China); Ovol1 (1:200) from Proteintech group (Chicago, IL, USA); cPLA2 α (1:1000) from GeneTex (Irvine, TX, USA); Phospho-cPLA2 α (1:1000), phospho-Akt (Ser473) (1:1000), phospho-FAK (1:1000), phospho-GSK-3 β (1:1000) Akt (Ser473)(1:1000), FAK (1:1000) and GSK-3 β (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and goat anti-rabbit, goat anti-mouse and donkey anti-goat secondary antibody from Santa Cruz Biotechnology (1:4000).

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Marker, Immunohistochemistry, Immunofluorescence