Journal: International Journal of Molecular Sciences

Article Title: Interleukin-34-CSF1R Signaling Axis Promotes Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/ijms22052711

Figure Lengend Snippet: Role of PIN1 in IL-34-induced MEK/ERK and JNK/c-Jun signaling pathways in JB6 Cl41 cells. ( A ) Cells were transfected with mock and Xpress-PIN1. At 24 h after transfection, the cells were serum starved for 24 h, treated with 10 ng/mL IL-34 for 15 min or left untreated, harvested, and lysed. The lysates were resolved using SDS-PAGE and immunoblotting analysis was performed using specific antibodies against corresponding proteins. ( B ) Cells were transfected with mouse siRNA-control and siRNA-PIN1. Cells were serum starved for 24 h, treated with 10 ng/mL IL-34 for 15 min or left untreated, harvested, and lysed. Proteins in whole cell lysates were separated using SDS-PAGE and immunoblotted. ( C ) Cells were serum starved for 24 h, pre-treated with the indicated concentrations of juglone for 12 h, and then exposed to 10 ng/mL IL-34 for 15 min, harvested, and lysed. Proteins in whole cell lysates were separated using SDS-PAGE and immunoblotted. Blots are representative of an experiment repeated at least three times with the similar result. The numbers below the band represent fold changes in protein levels after normalization to β-actin using densitometric quantification by ImageJ.

Article Snippet: The antibodies acquired from Santa Cruz Biotechnology (Dallas, TX, USA) were as follows: c-Fms/CSF1R mouse mAb (C-20) (#sc-692, detects both unprocessed and processed forms of c-Fms/CSF1R at 130 and 165 kDa, respectively), PIN1 (G-8) mouse mAb (#sc-46660, detects a specific band at 20 kDa), c-Jun mouse mAb (G-4) (#sc-74543, detects a specific band at 48 kDa).

Techniques: Protein-Protein interactions, Transfection, SDS Page, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-34-CSF1R Signaling Axis Promotes Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/ijms22052711

Figure Lengend Snippet: Involvement of PIN1 in IL-34-induced MAPK pathway in breast cancer cells. ( A , B ) SKBR3 and MCF7 cells were serum starved for 24 h, treated with the indicated doses of IL-34 for 30 min ( A ) or 10 ng/mL IL-34 for indicated times ( B ), harvested, and lysed. The lysates were resolved using SDS-PAGE and immunoblotting analysis was performed using specific antibodies against corresponding proteins. ( C – E ) MCF7 cells were transfected with siRNA-control and siRNA-CSF1R ( C ) mock and Xpress-PIN1 ( D ) or siRNA-control and siRNA-PIN1 ( E ), serum starved for 24 h and then treated with 10 ng/mL IL-34 for 15 min or left untreated, harvested, and lysed. Proteins in whole cell lysates were separated using SDS-PAGE and immunoblotted. ( F ) MCF7 cells were serum starved for 24 h, pre-treated with the indicated concentrations of juglone for 12 h, and then exposed to 10 ng/mL IL-34 for 15 min, harvested, and lysed. Proteins in whole cell lysates were separated using SDS-PAGE and immunoblotted. ( A – F ) Blots are representative of an experiment repeated at least three times with the similar result. The numbers below the band represent fold changes in protein levels after normalization to β-actin using densitometric quantification by ImageJ.

Article Snippet: The antibodies acquired from Santa Cruz Biotechnology (Dallas, TX, USA) were as follows: c-Fms/CSF1R mouse mAb (C-20) (#sc-692, detects both unprocessed and processed forms of c-Fms/CSF1R at 130 and 165 kDa, respectively), PIN1 (G-8) mouse mAb (#sc-46660, detects a specific band at 20 kDa), c-Jun mouse mAb (G-4) (#sc-74543, detects a specific band at 48 kDa).

Techniques: SDS Page, Western Blot, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-34-CSF1R Signaling Axis Promotes Epithelial Cell Transformation and Breast Tumorigenesis

doi: 10.3390/ijms22052711

Figure Lengend Snippet: Role of PIN1 in AP-1 activity and breast tumorigenesis induced by IL-34. MCF7 cells were seeded and co-transfected with the luciferase reporters, c-Fos-luc ( A ), c-Jun-luc ( B ), and AP-1-luc ( C ) along with the pRL-TK vector. At 24 h after transfection, the cells were serum starved for 24 h and then treated with the indicated doses of IL-34 for 24 h before luciferase assay was performed. ( D ) MCF7 cells were seeded and co-transfected with the luciferase reporter AP-1-luc and siRNA-control or AP-1-luc and siRNA-PIN1. At 24 h after transfection, the cells were serum starved for 24 h, treated with 10 ng/mL IL-34 for 24 h or left untreated, before a luciferase assay was performed. ( E ) MCF7 cells were transfected with siRNA-control and siRNA-PIN1. After 24 h, the cells were treated with 10 ng/mL IL-34 in a soft agar matrix or left untreated and incubated at 37 °C in a 5% CO 2 atmosphere. After 14 days, colonies from three separate experiments were photographed (left), followed by calculation of the average colony numbers and sizes (diameter > 200 μm, right). ( F ) 4T1 cells were injected into the mammary gland of BALB/c mice in the presence or absence of 100 ng/mL IL-34 and 100 μM juglone, and allowed to grow until tumors were formed. Shown are representative pictures of tumor (left), measured volumes and weights of tumors (right). Error bars indicate the mean ± S.D. of triplicate measurements from two independent experiments. Statistical analyses were conducted using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, compared to control group or only IL-34-treated group, respectively).

Article Snippet: The antibodies acquired from Santa Cruz Biotechnology (Dallas, TX, USA) were as follows: c-Fms/CSF1R mouse mAb (C-20) (#sc-692, detects both unprocessed and processed forms of c-Fms/CSF1R at 130 and 165 kDa, respectively), PIN1 (G-8) mouse mAb (#sc-46660, detects a specific band at 20 kDa), c-Jun mouse mAb (G-4) (#sc-74543, detects a specific band at 48 kDa).

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Control, Incubation, Injection