Journal: Molecular Medicine Reports

Article Title: Adenoviruses-mediated RNA interference targeting cytosolic phospholipase A2α attenuates focal ischemic brain damage in mice

doi: 10.3892/mmr.2018.8610

Figure Lengend Snippet: Effects of siRNAs for mouse cPLA2α on expression levels of cPLA2α in L929 cells. L929 cells were transfected with Ad-siRNA-cPLA2α (pSilencer nos. 1–3) or negative control, L929 cells were treated with 10 nM ATK every 24 h as a positive control. Mock group cells were treated with the same quantity of ATK solvent solution at the same time. (A) The protein expression levels of cPLA2α in the different groups, with β-actin serving as the control. (B) mRNA expression levels of cPLA2α. ***P<0.001 (Student's t-test). siRNA, small interfering; cPLA2α, cytosolic phospholipase A2α; ATK, arachidonyl trifluoromethyl ketone.

Article Snippet: The proteins (40 μg) were separated by 12% SDS-PAGE, then transferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat dried milk at 37°C for 2 h. Membranes were incubated with the following primary antibodies for 16 h at 4°C: Anti-cPLA2α antibody (cat. no. sc-137069; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin antibody (cat. no. sc-8432; 1:200; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Transfection, Negative Control, Positive Control, Solvent, Control

Journal: Molecular Medicine Reports

Article Title: Adenoviruses-mediated RNA interference targeting cytosolic phospholipase A2α attenuates focal ischemic brain damage in mice

doi: 10.3892/mmr.2018.8610

Figure Lengend Snippet: Effects of siRNA-cPLA2α on mouse blood flow prior to and following MCAO/R. Laser-Doppler flow measurement of cerebral blood flow was performed prior to surgery (0–20 min), during the surgery (20–80 min) and in the reperfusion (80–100 min) period. Data are presented as the mean ± standard deviation of the mice in the same group. pAd-siRNA-cPLA2α treatment does not alter relative cerebral blood flow. MCAO/R, middle cerebral artery occlusion and/or reperfusion; siRNA, small interfering RNA; cPLA2α, cytosolic phospholipase A2α.

Article Snippet: The proteins (40 μg) were separated by 12% SDS-PAGE, then transferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat dried milk at 37°C for 2 h. Membranes were incubated with the following primary antibodies for 16 h at 4°C: Anti-cPLA2α antibody (cat. no. sc-137069; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin antibody (cat. no. sc-8432; 1:200; Santa Cruz Biotechnology, Inc.).

Techniques: Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Adenoviruses-mediated RNA interference targeting cytosolic phospholipase A2α attenuates focal ischemic brain damage in mice

doi: 10.3892/mmr.2018.8610

Figure Lengend Snippet: Expression levels of cPLA2α in mice brains following MCAO/R. (A) Representative western blot analysis of cPLA2α protein levels in whole brain lysates from the stroke brains of mice at 2 h, day 1, 3, 7 and 14 after the MCAO/R procedure. (B and C) Expression levels of cPLA2α mRNA at different time-points following MCAO/R surgery were detected by quantitative polymerase chain reaction. Data are presented as means ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001 (Student's t-test). cPLA2α, cytosolic phospholipase A2α; MCAO/R, middle cerebral artery occlusion and/or reperfusion; NC, negative control; RNAi, RNA interference.

Article Snippet: The proteins (40 μg) were separated by 12% SDS-PAGE, then transferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat dried milk at 37°C for 2 h. Membranes were incubated with the following primary antibodies for 16 h at 4°C: Anti-cPLA2α antibody (cat. no. sc-137069; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin antibody (cat. no. sc-8432; 1:200; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Negative Control

Journal: Molecular Medicine Reports

Article Title: Adenoviruses-mediated RNA interference targeting cytosolic phospholipase A2α attenuates focal ischemic brain damage in mice

doi: 10.3892/mmr.2018.8610

Figure Lengend Snippet: Effects of siRNA-cPLA2α on the expression of FFA, LPC, PGE2 and LTB4 in mice after MCAO/R. Expression levels of (A) FFA and (B) LPC in the brain tissue from mice 24 h after MCAO/R surgery were measured by high performance thin layer chromatography. Expression levels of (C) PGE2 and (D) LTB4 in the serum of mice 24 h after MCAO/R surgery were measured using ELISA. pAd-siRNA-cPLA2α treatment decreased the expression levels of FFA, LPC, PGE2 and LTB4. Data are presented as means ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001 (Student's t-test). siRNA, small interfering RNA; cPLA2α, cytosolic phospholipase A2α; FFA, free fatty acids; LPC, lysophosphatidylcholine; PGE2, prostaglandin E2; LTB4, leukotriene B4; MCAO/R, middle cerebral artery occlusion and/or reperfusion; NC, negative control.

Article Snippet: The proteins (40 μg) were separated by 12% SDS-PAGE, then transferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat dried milk at 37°C for 2 h. Membranes were incubated with the following primary antibodies for 16 h at 4°C: Anti-cPLA2α antibody (cat. no. sc-137069; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin antibody (cat. no. sc-8432; 1:200; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, High Performance Thin Layer Chromatography, Enzyme-linked Immunosorbent Assay, Standard Deviation, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: Adenoviruses-mediated RNA interference targeting cytosolic phospholipase A2α attenuates focal ischemic brain damage in mice

doi: 10.3892/mmr.2018.8610

Figure Lengend Snippet: Effects of siRNA-cPLA2α on pathological changes, cell apoptosis, and infarct volume of mice following MCAO/R. (A) Pathological changes of hippocampal and cortical tissues following pAd-siRNA-cPLA2α treatment (magnification, ×100). (B) PAd-siRNA-cPLA2α treatment protects mice from brain infarction 14 days after MCAO/R. (C) Representative images of TUNEL staining from mice brain tissue samples 14 days after MCAO/R (magnification, ×200). (D) Triphenyltetrazolium chloride staining images from mice brain tissue samples 14 days after MCAO/R. (E) Number of TUNEL-positive cells was counted. Data are presented as means ± standard deviation of three independent experiments. *P<0.05 and **P<0.01, RNAi group vs. the MCAO/R and NC group; ### P<0.001, sham group vs. the other three groups (Student's t-test). siRNA, small interfering RNA; cPLA2α, cytosolic phospholipase A2α; MCAO/R, middle cerebral artery occlusion and/or reperfusion; RNAi, RNA interference; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labeling; NC, negative control.

Article Snippet: The proteins (40 μg) were separated by 12% SDS-PAGE, then transferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat dried milk at 37°C for 2 h. Membranes were incubated with the following primary antibodies for 16 h at 4°C: Anti-cPLA2α antibody (cat. no. sc-137069; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin antibody (cat. no. sc-8432; 1:200; Santa Cruz Biotechnology, Inc.).

Techniques: TUNEL Assay, Staining, Standard Deviation, Small Interfering RNA, Negative Control

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

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