Journal: Journal of Cellular and Molecular Medicine

Article Title: Candidate tumour suppressor CCDC19 regulates miR-184 direct targeting of C-Myc thereby suppressing cell growth in non-small cell lung cancers

doi: 10.1111/jcmm.12317

Figure Lengend Snippet: CCDC19 controls the expression of cell cycle signals and transcription factor C-JUN in NSCLC via PI3K/AKT pathway. ( A ) Overexpressed CCDC19 suppressed the expression of pPI3K and p-AKT, but not their total protein levels in CCDC19-overexpressing A549 and SPCA1 cells compared to their respective mock cells. ( B ) Restored CCDC19 inhibited the expression of C-Myc, CCND1, E2F1 and C-JUN and induced the expression of p21, p27, p15 and p16 in A549 and SPCA1 cells. Increased expression of E-Cadhein was only observed in CCDC19-overexpressed A549 cells. SPCA1 cells did not express E-Cadherin. ( C ) Knocking down CCDC19 by siRNA restored the activation of pPI3K and pAKT, stimulated the expression of C-Myc and CCND1, as well as repressed p15 and p21 expression in CCDC19-overexpressed A549 and SPCA1 cells. Reduced expression of E-Cadherin was only observed in CCDC19-suppressed A549 cells. SPCA1 cells did not express E-Cadherin.

Article Snippet: Western blotting was performed with a SDS-PAGE Electrophoresis System according to previous reports [ , ] with the following antibodies: rabbit polyclonal CCDC19 (Proteintech Group, Inc. Chicago, IL, USA), ACTB, GAPDH, CCND1, p21, E2F1, p15 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), C-Myc, C-Jun, AKT, p-Akt (Ser-473), PI3K, p16, p27, E-Cadherin and pPI3K (Tyr458; 1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Candidate tumour suppressor CCDC19 regulates miR-184 direct targeting of C-Myc thereby suppressing cell growth in non-small cell lung cancers

doi: 10.1111/jcmm.12317

Figure Lengend Snippet: miR-184 directly targets C-MYC-mediated cell cycle signal suppressing cell growth in NSCLC. ( A ) The introduction of miR-184 mimics suppressed cell growth in lung cancer A549 and SPCA1 cells by MTT assay. ( B ) Transfection of miR-184 inhibitor induced cell proliferation in A549 and SPCA1 lung cancer cells by MTT assay. ( C ) The introduction of miR-184 mimics suppressed the expression of C-Myc and CCND1 and stimulated p21 and p15 expression in A549 and SPCA1 cells. ( D ) miR-184 inhibitor suppressed the expression C-Myc in A549 and SPCA1 cells. ( E ) Luciferase reporter assay was used to determine miR-184 direct targeting of the C-Myc coding region. Data are presented as mean ± SD for three independent experiments (* P < 0.05).

Article Snippet: Western blotting was performed with a SDS-PAGE Electrophoresis System according to previous reports [ , ] with the following antibodies: rabbit polyclonal CCDC19 (Proteintech Group, Inc. Chicago, IL, USA), ACTB, GAPDH, CCND1, p21, E2F1, p15 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), C-Myc, C-Jun, AKT, p-Akt (Ser-473), PI3K, p16, p27, E-Cadherin and pPI3K (Tyr458; 1:1000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: MTT Assay, Transfection, Expressing, Luciferase, Reporter Assay

Journal: Oncotarget

Article Title: GPER is involved in the regulation of the estrogen-metabolizing CYP1B1 enzyme in breast cancer.

doi: 10.18632/oncotarget.22541

Figure Lengend Snippet: Figure 4: GPER and CYP1B1 mediate the up-regulation of cyclins D1, cyclin E and cyclin A by E2 and G-1 in SkBr3 cells, CAFs and met-CAFs. Cyclin D1, cyclin E and cyclin A mRNA expression in SkBr3 cells (A), CAFs (C) and met-CAFs (E) treated for 18 h with vehicle (−), E2 (10 nM) and G-1 (100 nM) alone or in combination with 100 nM GPER antagonist G15 and 5 μM CYP1B1 inhibitor TMS, as evaluated by real-time PCR. Data obtained in three independent experiments performed in triplicate were normalized to 18S expression and shown as fold changes upon E2 and G-1 treatments respect to cells treated with vehicle. (■) P < 0.05 for cells receiving treatments versus vehicle. Cyclin D1, cyclin E and cyclin A protein levels in SkBr3 cells (B), CAFs (D) and met-CAFs (F) upon treatments for 18 h with vehicle (−), E2 (10 nM) and G-1 (100 nM) alone or in combination with 100 nM GPER antagonist G15 and 5 μM CYP1B1 inhibitor TMS. β-actin serves as a loading control. Results shown are representative of at least two independent experiments.

Article Snippet: Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Adrich, Milan, Italy), probed overnight at 4°C with antibodies against CYP1B1 (TA339934), cyclin D1 (TA801655), cyclin E (TA590076), cyclin A (TA890057) (OriGene Technologies, DBA, Milan, Italy), c-Fos (E8), GPER (N-15) and β-actin (C-2) (Santa Cruz Biotechnology, DBA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Oncotarget

Article Title: GPER is involved in the regulation of the estrogen-metabolizing CYP1B1 enzyme in breast cancer.

doi: 10.18632/oncotarget.22541

Figure Lengend Snippet: Figure 6: E2 and G-1 induce CYP1B1 expression through GPER in MDA-MB-231 breast cancer cells. (A) E2 (10 nM) and G-1 (100 nM) induce CYP1B1 mRNA expression in MDA-MB-231 cells, as evaluated by real-time PCR. Data obtained in three independent experiments performed in triplicate were normalized to 18S expression and shown as fold changes upon E2 and G-1 treatments respect to cells exposed to vehicle (−). (B–C) CYP1B1 protein levels in MDA-MB-231 cells treated with 10 nM E2 and 100 nM G-1, as indicated. (D) CYP1B1 protein levels upon treatments with 10 nM E2 and 100 nM G-1 in cells transfected with shRNA or shGPER. (E) Efficacy of GPER silencing. β-actin serves as a loading control. Results shown are representative of at least two independent experiments. (F) Cells were transiently transfected for 8 h with the indicated CYP1B1 promoter constructs, then cells were treated for 18 h with vehicle (−), 10 nM E2 or 100 nM G-1. (G) Cells were transiently transfected for 8 h with the deleted CYP1B1 promoter constructs shown in Figure 2C and 2D, then treated for 18 h with vehicle, 10 nM E2 and 100 nM G-1, as indicated. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle were set as 1-fold induction upon which the activities induced by treatments were calculated. Each column represents the mean ± SD for three independent experiments, each performed in triplicate. (H) Cyclin D1, cyclin E and cyclin A protein levels in cells transiently transfected with a shRNA or shGPER for 24 h, then

Article Snippet: Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Adrich, Milan, Italy), probed overnight at 4°C with antibodies against CYP1B1 (TA339934), cyclin D1 (TA801655), cyclin E (TA590076), cyclin A (TA890057) (OriGene Technologies, DBA, Milan, Italy), c-Fos (E8), GPER (N-15) and β-actin (C-2) (Santa Cruz Biotechnology, DBA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, shRNA, Control, Construct, Luciferase

Journal: Oncotarget

Article Title: GPER is involved in the regulation of the estrogen-metabolizing CYP1B1 enzyme in breast cancer.

doi: 10.18632/oncotarget.22541

Figure Lengend Snippet: Figure 7: CYP1B1 is involved in the growth of MDA-MB-231 xenografts. (A) Tumor volume from MDA-MB-231 xenografts implanted in female athymic nude mice treated for 21 days with vehicle, G-1, TMS or both compounds, as indicated. (*) indicates P < 0.05 for animals treated with G-1 versus animals treated with vehicle. (B) Representative images of explanted tumors at day 21, scale bar, 0.3 cm. (C) Cyclin D1, cyclin E, cyclin A protein levels in tumor homogenates from MDA-MB-231 xenografts treated as reported above. β-actin serves as loading control. Results shown are representative of two independent experiments. (D) Ki67, cyclin D1, cyclin E and cyclin A immunodetection in paraffin embedded sections of explanted tumors from breast cancer xenografts treated with vehicle, G-1 and TMS alone or in combination, as indicated. Scale bar: 25 μm. Insert: negative control. Histograms represent the percentage (± SD) of immunostained positive cells treated with G-1 and TMS alone or in combination versus vehicle treated cells. (*) indicates P < 0.05.

Article Snippet: Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Sigma-Adrich, Milan, Italy), probed overnight at 4°C with antibodies against CYP1B1 (TA339934), cyclin D1 (TA801655), cyclin E (TA590076), cyclin A (TA890057) (OriGene Technologies, DBA, Milan, Italy), c-Fos (E8), GPER (N-15) and β-actin (C-2) (Santa Cruz Biotechnology, DBA).

Techniques: Control, Immunodetection, Negative Control

Journal: Bioengineered

Article Title: Bone mesenchymal stem cells-derived miR-223-3p-containing exosomes ameliorate lipopolysaccharide-induced acute uterine injury via interacting with endothelial progenitor cells

doi: 10.1080/21655979.2021.2001185

Figure Lengend Snippet: LPS suppressed EPCs cell viability via inducing cell pyroptosis. (a, b) NLRP3 was silenced in EPCs cells. (c) MTT assay was used to examine cell viability. (d) The expression status of Cyclin D1 and CDK2 were determined by Western blot analysis. Individual experiment repeated 3 times, and * P < 0.05

Article Snippet: Next, the PVDF membranes were incubated with the primary antibodies against NLRP3 (1:1500, Catalog MAB7578, R&D system, USA), ASC (1:1000, Catalog AF3805, R&D system, USA), N-Gasdermin D (1:1000, Catalog ab209845, Abcam, UK), GAPDH (1:2000, Catalog 2275-PC-100, R&D system, USA), cleaved Caspase-3 (1:1500, Catalog AF835, R&D system, USA), Bax (1:2000, AF820, R&D system, USA), Cyclin D1 (1:1500, Catalog MAB4314, R&D system, USA) and CDK2 (1:2000, Catalog AF4654, R&D system, USA) at 4 °C overnight.

Techniques: MTT Assay, Expressing, Western Blot